p27基因和中药“片仔癀”抑制骨肉瘤生长的实验研究
发布时间：2018-07-31 14:25
【摘要】：实验背景及目的 骨肉瘤(Osteosarcoma)亦称为成骨肉瘤,是高度恶性的原发性骨肿瘤,发病年龄在10-30岁之间,发病率居原发性恶性肿瘤之首。骨肉瘤的病程进展很快,在临床确诊病例中,80%的患者已发现有血行远隔转移。经过手术前动脉灌注化疗结合术后化疗,能够让多数患者避免截肢,明显提高了生存质量,预后有所改善,生存率有了明显提高。但是,骨肉瘤较高的侵袭转移能力以及对化疗药物的耐药,使得部分患者在接受了规范手术和化疗以后,仍然出现早期复发或早期转移。p27是调节细胞周期G1/S期的一种抑制因子,在维持细胞周期顺利进行中起关键性作用。体外实验研究发现,作为一个细胞周期抑制因子,p27不仅能抑制正常的血管内皮细胞增殖和迁移,也能诱导细胞凋亡。实验发现p27在体内能够抑制各种原发性肿瘤和转移性肿瘤的生长,p27基因的缺失是导致骨肉瘤的发生和病变进展的重要原因之一。根据报道,p27基因在人类骨肉瘤移植瘤裸鼠模型中,可以改善肿瘤裸鼠的预后,缓解症状,延长生存时间。中药“片仔癀”具有清热解毒,减少肿胀和软坚散结的功效,能够促进血液循环,消除血瘀,临床多用于消化系统肿瘤的治疗以及防治肿瘤化疗过程中的肝损害。有研究证实片仔癀对骨肉瘤移植瘤细胞具有诱导凋亡的作用。文献报告人类骨肉瘤细胞株MG63、 Saos-2等,经细胞培养技术移植于裸鼠骨髓腔内可形成骨肉瘤病理模型；而腺相关病毒可作为p27基因载体,并在动物体内表达p27蛋白,且具有相关生物活性。因此,本实验选取人类骨肉瘤Saos-2细胞株,建立裸鼠原位移植瘤模型,使用腺相关病毒作为p27基因载体,分别观察和比较p27基因、中药“片仔癀”以及两者联合应用对人类骨肉瘤生长的抑制作用,为以后骨肉瘤的基因治疗以及中药治疗提供实验依据。 实验材料 1、细胞、药物及试剂：人类骨肉瘤Saos-2细胞系,购于上海超研生物科技有限公司；片仔癀(主要成分含有麝香、牛黄、三七、蛇胆等),购于漳州片仔癀制药有限公司(生产批号：H.M.L.N.Z20080011);转染raav-p27的重组腺相关病毒溶液、空载体重组腺相关病毒(rAAV-MCS)悬液,购于西安华光生物工程公司；胎牛血清(FBS),购于HyClone公司(NE, USA), p27抗体,购于上海宝特生命科学发展有限公司；酶链亲和素-生物素(SABC)试剂盒,购于上海复中生物技术有限公司；兔抗β-actin抗体,美国Santa Cruze公司产品,辣根过氧化物酶标记的羊抗兔二抗,美国Santa Cruze公司产品；ECL试剂盒,美国Santa Cruze公司产品；BCA蛋白浓度测定试剂盒,美国Bio-Rad公司产品。 2、主要仪器设备：血红细胞计数器(上海实验室仪器有限公司,中国);半干转印仪(Semidry Transfer system,美国Bio-Rad公司)；紫外凝胶自动成像仪(上海勤翔科学仪器有限公司,中国) 实验方法 1、实验动物： BALB/C裸鼠30只,SPF级无特殊病原体,3-4周龄,体重15-20g,雌雄不限,规格为：CAnN.Cg-Foxn1nu/Cr1VR,购自上海宝特生命科学发展有限公司。饲养条件：恒温25-27℃,湿度45%-50%,半屏障系统环境下饲养,食用灭菌处理过的饲料、水,高效过滤系统处理过的空气,每小时换气10-15次,一天10小时照明,14小时在无照明黑暗条件下饲养。 2、人类骨肉瘤Saos-2细胞株的细胞培养： 细胞培养液的制备：采用Roswell Park Memorial Institute (RPMI)-1640培养液,加入10%标准胎牛血清,2mmol/L-谷氨酰铵,100U/ml青霉素和0.1mg/ml链霉素,过滤和消毒后,4℃储存备用。将装有人类骨肉瘤Saos-2细胞株的冻存管从液氮罐里取出,解冻、洗涤、离心处理后,将细胞悬液转移至10ml培养皿中,放置在37℃,5%CO2培养箱中过夜。当细胞生长至相对密度80%左右时,加入胰酶消化液,在37℃,5%的CO2培养箱里消化2-3分钟,然后加入2ml细胞培养液终止细胞消化,此时,在显微镜下可观察到大多数的游离圆形细胞。用吸管将细胞充分搅匀后,静置。离心弃上清,加入5ml细胞培养液,吹打均匀。用细胞计数器计数后备用。 3、在无菌条件下,用4%水合氯醛(10μ L/g)将裸鼠麻醉。然后用1ml规格注射器4号针头吸取0.1ml细胞悬液(1×107个细胞／注射器)注射到裸鼠右侧腋窝皮下,每次注射两只裸鼠。2-4周后,待观察到裸鼠皮下移植瘤生长至1.0cm×1.0cm×1.5cm大小后,处死裸鼠,在无菌条件下取除骨肉瘤瘤体组织。将得到的腋窝肿瘤组织处理成0.1cm×0.1cm×0.1cm大小后再移植在新的裸鼠右后腿上端胫骨骨髓腔内,然后将裸鼠放回饲养笼,观察肿瘤的生长。此时,人类骨肉瘤Saos-2细胞株裸鼠原位模型建立成功。肿瘤细胞接种后,动物生长状况良好,在接种Saos-2细胞株5天之后,可见肿瘤组织在裸鼠皮下已随时间增长。接种六周后,没有出现动物死亡,肿瘤相对体积增长率为92.7%。 4、实验分组及处理： 肿瘤细胞接种一周后,待肿瘤生长至0.8cm时,挑选30只肿瘤体积相近的裸鼠按随机数字表随机分为5组,每组6只： 空白对照组,用100μLPBS注射肿瘤部位,3天1次； 空载体对照组,用100μL多克隆位点重组腺相关病毒(rAAV-MCS)悬液注射肿瘤部位,空载体病毒溶液浓度1×1011/L,3天1次； 片仔癀药物组,在肿瘤形成后,按0.01ml/g剂量,每天早晚各喂食给药一次,共两次给药。 p27基因组,用100μL raav-p27病毒溶液注射,病毒溶液浓度1×1011/L注射肿瘤部位,3天1次。 5.联合治疗组,用100μL raav-p27病毒溶液注射肿瘤部位,3天1次,同时喂食片仔癀溶液,一天早晚两次给药。 在裸鼠接种之后,观察并记录它们的精神状态,体重,食欲、粪便和尿液。如果实验中发现裸鼠死亡,解剖并分析死亡原因。 肿瘤细胞接种6周之后,用颈椎脱臼法将所有裸鼠处死。剥离肿瘤组织,称重并记录肿瘤重量,计算肿瘤生长抑制率,计算公式为：(对照组瘤重-实验组瘤重)／对照组瘤重×100%。在动物模型建立之后,用游标卡尺测量每组裸鼠的第1,4,8,12,16,18和22天肿瘤的最大和最小直径,并应用Steel公式V=1/2ab2计算体积,其中a,b分别是肿瘤的最大和最小直径。 5、免疫组织化学染色：将肿瘤组织用福尔马林固定,石蜡包埋后进行免疫组织化学方法(IHC)染色。使用鼠抗p27抗体(一抗)及碱性磷酸酶标记的马抗鼠IgG(二抗),运用链霉亲和素-生物素复合物(SABC法),并运用双重染色法检测p27的蛋白表达。使用光学显微镜观察,在放大10倍的视野内统计阳性细胞表达率,来判断p27的表达情况。 6、蛋白质印迹分析：从肿瘤组织细胞中提取蛋白,然后用抗p27兔单抗和兔抗β-actin内参抗体来进行免疫印迹实验(Western-Blot)。经ECL化学发光底物曝光后,用实验室图像分析软件计算蛋白含量。 7、统计分析：采用SPSS10.0分析软件程序来进行统计分析。数据采用平均值±标准差(X±S),与对比组进行单向方差分析和LSD分析。 结果 1、动物模型的症状观察：及肿瘤细胞移植到裸鼠体内7周之后,p27基因组,联合治疗组以及片仔癀药物组所有的裸鼠都存活下来,空白对照组和空载体对照组各有1只死亡。空白对照组和空载体对照组的裸鼠肿瘤体积增长非常明显,且两者之间没有显著差异,裸鼠的精神状态和食欲下降,尿液颜色偏暗,粪便偏干,体重下降。片仔癀药物组的裸鼠肿瘤增长相对较缓慢,精神状态良好,食欲正常,体重变化不大,粪便和尿液的颜色和性状均正常。p27基因组的裸鼠肿瘤增长同样较慢,精神状态一般,食欲略有下降,体重随着肿瘤体积增加有所增长,但体型偏瘦,偶尔还存在腹泻。联合治疗组的肿瘤生长抑制明显,裸鼠的精神状态很好,饮食、粪便和尿液正常,没有腹泻现象,但体重略有下降。 2、肿瘤生长情况和抑制率统计：空白对照组与空载体对照组的肿瘤体积相近(P0.05)。在第8,12,16,18和22天记录的肿瘤体比较中,p27基因组和片仔癀药物组与空白对照组相比,呈明显下降(P0.05)；联合治疗组与片仔癀药物组或者p27基因组相比,也是明显减少(P0.05)。在肿瘤平均重量比较上,片仔癀药物组和p27基因组比空白对照组轻(P0.05)。联合治疗组比片仔癀药物组或者p27基因组也要轻(P0.05)。因此,可判断在肿瘤抑制率上,片仔癀药物组和p27基因组比空白对照组高(P0.05)。联合治疗组比片仔癀药物组或者p27基因组同样也高(P0.05)。 3、p27蛋白表达情况：经染色后的p27蛋白阳性细胞,可在细胞核里观察到棕黄色颗粒。在p27蛋白阳性率统计上,p27基因组比空白对照组有显著提高(P0.05),联合治疗组比p27基因组或者片仔癀药物组也有明显提高(P0.05)。免疫印迹试验显示在p27基因表达量比较上,p27基因组比空白对照组高(P0.05),联合治疗组比p27基因组或者片仔癀高(P0.05) 结论 1、Saos-2人类骨肉瘤细胞株经培养传代后可移植裸鼠获得肿瘤原位生长的动物模型。 2、腺相关病毒作为基因载体对肿瘤生长无影响,转染p27基因的腺相关病毒可增加肿瘤组织内p27蛋白的表达。 3、p27基因、片仔癀均有抑制骨肉瘤生长的作用,二者联合应用时对肿瘤生长的抑制作用明显增强。 意义 证实了p27重组腺相关病毒可用于骨肉瘤的研究和治疗,p27基因与中药片仔癀联合应用对骨肉瘤的抑制有协同作用。初步探讨了腺相关病毒作为基因载体用于肿瘤基因治疗的可能性,在骨肉瘤的临床治疗方面,为p27基因联合中药片仔癀的疗效研究,进一步奠定了理论基础。
[Abstract]:Experimental background and purpose
Osteosarcoma (Osteosarcoma), also known as osteosarcoma, is a highly malignant primary bone tumor with a incidence of 10-30 years of age. The incidence of osteosarcoma is the first of primary malignant tumors. The course of osteosarcoma is progressing rapidly. In clinical cases, 80% of the patients have been found to have distant metastasis. Treatment, which allows most patients to avoid amputation, significantly improves the quality of life, improves the prognosis, and has a significant improvement in survival. However, the high invasion and metastasis ability of osteosarcoma and the resistance to chemotherapeutic drugs make some patients still have early recurrence or early metastasis.P27 after receiving standardized surgery and chemotherapy. A inhibitory factor in the cell cycle G1/S stage plays a key role in maintaining a smooth cell cycle. In vitro studies have found that, as a cell cycle inhibitor, p27 can not only inhibit the proliferation and migration of normal vascular endothelial cells, but also induce apoptosis. It is found that p27 can inhibit the primary swelling in the body. The growth of tumor and metastatic tumor, the deletion of p27 gene is one of the important reasons for the occurrence and progression of osteosarcoma. According to the report, the p27 gene in the nude mice model of human osteosarcoma transplanted tumor can improve the prognosis, relieve symptoms and prolong the survival time of tumor nude mice. The effect of soft hard and loose knot can promote blood circulation, eliminate blood stasis, use the treatment of digestive system tumor and prevent the liver damage in the process of tumor chemotherapy. The pathological model of osteosarcoma can be formed in the bone marrow cavity of nude mice. Adeno-related virus can be used as a carrier of p27 gene and expression of p27 protein in animals, and it has related biological activity. Therefore, this experiment selected human osteosarcoma Saos-2 cell lines, established the orthotopic xenograft model in nude mice, and used adeno-related virus as the p27 gene carrier. The inhibitory effects of p27 gene, Chinese herbal medicine and the combination of them on the growth of human osteosarcoma were observed and compared, and the experimental basis was provided for the gene therapy of osteosarcoma and the treatment of traditional Chinese medicine.
Experimental materials
1, cells, drugs and reagents: human osteosarcoma Saos-2 cell line, purchased in Shanghai Chao Yan Biological Technology Co., Ltd. (mainly composed of musk, bezoar, 37, snake gall), purchased in Zhangzhou Zai Zai Jin Pharmaceutical Co., Ltd. (production batch number: H.M.L.N.Z20080011); recombinant adeno-associated virus solution transfected with raav-p27, unloaded body weight group Adeno-related virus (rAAV-MCS) suspension, purchased in Xi'an Huaguang bioengineering company; fetal bovine serum (FBS), purchased in HyClone (NE, USA), p27 antibody, purchased in Shanghai Life Science Development Co., Ltd., enzyme chain avidin biotin (SABC) kit, purchased in Shanghai compound Biotechnology Co., Ltd., Rabbit anti beta -actin antibody, American Santa Cruze company products, horseradish peroxidase labelled Sheep anti rabbit two resistance, American Santa Cruze company products; ECL kit, Santa Cruze company products in the United States; BCA protein concentration test kit, American Bio-Rad company product.
2, the main instrument and equipment: blood red cell counter (Shanghai Laboratory Instrument Co., Ltd., China); semi dry transfer printer (Semidry Transfer system, American Bio-Rad company); ultraviolet gel automatic imaging instrument (Shanghai Qin Xiang Science Instrument Co., Ltd., China)
Experimental method
1, experimental animals:
BALB/C nude mice 30, SPF class no special pathogens, 3-4 weeks old, weight 15-20g, male and female, the specification is: CAnN.Cg-Foxn1nu/Cr1VR, purchased from Shanghai Bao Life Science Development Co., Ltd.. Feeding conditions: constant temperature 25-27, humidity 45%-50%, half barrier system environment, food, water, efficient filtration system treated The air is ventilated 10-15 times per hour, 10 hours a day, and 14 hours under dark conditions without lighting.
2, the cell culture of human osteosarcoma Saos-2 cell line:
Preparation of cell culture solution: using Roswell Park Memorial Institute (RPMI) -1640 culture solution, adding 10% standard fetal bovine serum, 2mmol/L- glutamyl ammonium, 100U/ml penicillin and 0.1mg/ml streptomycin, after filtration and disinfection, stored at 4 C. The cryopreservation tube containing human osteosarcoma Saos-2 cell line is removed, thawing, washing, and away from the liquid nitrogen tank. After the heart treatment, the cell suspension was transferred to the 10ml culture dish and placed in the 5%CO2 culture box for the night. When the cells grew to the relative density of about 80%, the cells were added to the digestive juice, digested at 37, 5% CO2 incubator for 2-3 minutes, and then added to the 2ml cell culture to terminate the cell digestion. At this time, the majority of the cells could be observed under the microscope. Free round cells. Stir the cells thoroughly with a straw and set aside. Discard the supernatant by centrifugation, add 5 ml cell culture medium and blow evenly. Count the cells with a cell counter and reserve them.
3, under aseptic conditions, the nude mice were anesthetized with 4% chloral chloral (10 L/g). Then the 0.1ml cell suspension (1 x 107 cells / syringes) was injected into the right armpit of the nude mice with the 1ml specification syringe needle 4 needle. After each injection of two nude mice for.2-4 weeks, it was observed that the subcutaneous transplanted tumor of the nude mice was grown to 1.0cm * 1.0cm x 1.5cm, and was executed. In nude mice, the osteosarcoma tissue was removed under the aseptic condition. After the axillary tumor tissue was treated as 0.1cm x 0.1cm x 0.1cm, the bone marrow cavity of the right hind leg of the nude mice was retransplanted into the bone marrow cavity of the right hind leg of the nude mice. Then the nude mice were put back in the cage to observe the growth of the tumor. At this time, the human osteosarcoma Saos-2 cell line in nude mice was established successfully. After inoculation of the tumor cells, the animals grew well. After 5 days of inoculation of the Saos-2 cell line, the tumor tissue had been growing subcutaneously in the nude mice. After six weeks of inoculation, no animal death was found, and the relative volume growth rate of the tumor was 92.7%.
4, experimental grouping and processing:
Thirty nude mice with similar tumor volume were randomly divided into five groups at the time of tumor growth of 0.8 cm after one week of inoculation.
In blank control group, tumor sites were injected with 100 micron LPBS for 1 days in 3 days.
In empty vector control group, the tumor site was injected with 100 mu L polyclonal site recombinant adeno-associated virus (rAAV-MCS) suspension. The concentration of empty vector virus solution was 1 *1011/L, once every 3 days.
After the tumor was formed, the drug group was given 0.01ml/g dose once a day, two times a day.
The p27 genome was injected with 100 L raav-p27 virus solution. The concentration of virus solution was 1 x 1011/L injection site, 3 days 1 times.
5. In the combined treatment group, the tumor site was injected with 100 mu L raav-p27 virus solution once every 3 days, and the tablets were fed with gall solution twice a day.
After inoculation in the nude mice, the mental state, weight, appetite, feces and urine were observed and recorded. The death of nude mice was found in the experiment and the cause of death was dissected and analyzed.
After 6 weeks of inoculation of tumor cells, all nude mice were killed by dislocated cervical vertebra. The tumor tissue was stripped, weighed and recorded, and the tumor growth inhibition rate was recorded. The formula was as follows: (control group tumor weight experimental group tumor weight) / control group of tumor weight x 100%. was established by using vernier caliper to measure the 1,4,8,12,16 of nude mice in each group. On the 18th and 22nd days, the maximum and minimum diameters of the tumor were calculated by Steel's formula V=1/2ab2, where a and B were the maximum and minimum diameters of the tumor, respectively.
5, immunohistochemical staining: the tumor tissue was immobilized with formalin, and the paraffin was embedded in the immunohistochemical staining method (IHC). The IgG (two anti) of the horse anti rat rat was labeled with anti p27 antibody (one antibody) and alkaline phosphatase, and the streptomycin biotin complex (SABC method) was used. The protein expression of p27 was detected by double staining. The expression of p27 was measured by optical microscopy, and the expression of positive cells was counted in a 10 fold field of view.
6, Western blot analysis: extract protein from tumor tissue cells, and then use anti p27 rabbit monoclonal antibody and Rabbit anti beta -actin internal reference antibody to carry out immunoblotting test (Western-Blot). After exposure to ECL chemiluminescence substrate, the protein content was calculated by laboratory image analysis software.
7, statistical analysis: the SPSS10.0 analysis software program was used to carry out statistical analysis. The data used mean mean standard deviation (X + S), and the one-way ANOVA and LSD analysis were carried out with the contrast group.
Result
1, the observation of the animal model: after the tumor cells were transplanted into the nude mice for 7 weeks, the p27 genome, the combined treatment group and all the nude mice were alive, with 1 deaths in the blank control group and the empty carrier control group. The growth of the tumor volume in the blank control group and the empty carrier group was very obvious, and two There was no significant difference in the mental state and appetite of the nude mice, the dark color of the urine, the dry feces and the loss of weight. The tumor growth of the nude mice was relatively slow, the mental state was good, the appetite was normal, the body weight changed little, the color of the feces and urine and the characters of the nude mice in the normal.P27 genome were the same. Slow, general mental state, a slight decline in appetite, weight increase with the increase of tumor volume, but the body is thin and occasional diarrhea. The tumor growth inhibition in the combined treatment group is obvious, the mental state of the nude mice is very good, the diet, feces and urine are normal, there is no diarrhea phenomenon, but the weight has a slight decline.
2, the tumor growth and inhibition rate: the volume of tumor in the blank control group and the empty vector control group was similar (P0.05). In the comparison of the tumor bodies recorded on the 8,12,16,18 and the 22 days, the p27 genome and the tablet group were significantly lower than those in the blank control group (P0.05). In comparison with the average weight of the tumor (P0.05), the drug group and the p27 genome were lighter than those in the blank control group (P0.05). The combined treatment group was lighter than that of the drug group or the p27 genome (P0.05). Therefore, in the tumor inhibition rate, the drug group and the p27 genome were higher than the blank control group (P0.05). The combined treatment group was also higher than that of pizin medicine group or p27 genome (P0.05).
3, p27 protein expression: after dyed p27 protein positive cells, the brown yellow granules can be observed in the nucleus. In the positive rate of p27 protein, the p27 genome is significantly higher than that in the blank control group (P0.05). The combined treatment group is also significantly higher than the p27 genome or the tablet group (P0.05). The Western blot test shows that in P2, the immunoblotting test shows P2 Compared with the blank control group, p27 genome was higher (P 0.05), and the combined treatment group was higher than p27 genome or litter gall (P 0.05).
conclusion
1, Saos-2 human osteosarcoma cell line can be transplanted into nude mice to obtain an animal model of tumor growth in situ.
2. Adeno-associated virus as a gene vector has no effect on tumor growth. Adeno-associated virus transfected with p27 gene can increase the expression of p27 protein in tumor tissue.
3. Both p27 gene and Tablet gall can inhibit the growth of osteosarcoma, and the inhibitory effect on the growth of osteosarcoma is obviously enhanced when they are combined.
Significance
The recombinant adeno-associated virus (p27) can be used in the study and treatment of osteosarcoma, and the combination of p27 gene and traditional Chinese medicine has synergistic effect on the inhibition of osteosarcoma. The possibility of adeno-related virus as a gene carrier for tumor gene therapy is preliminarily discussed. In the treatment of osteosarcoma, the p27 gene is combined with Chinese herbal medicine. The research of curative effect has laid the theoretical foundation further.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R738.1
[Abstract]:Experimental background and purpose
Osteosarcoma (Osteosarcoma), also known as osteosarcoma, is a highly malignant primary bone tumor with a incidence of 10-30 years of age. The incidence of osteosarcoma is the first of primary malignant tumors. The course of osteosarcoma is progressing rapidly. In clinical cases, 80% of the patients have been found to have distant metastasis. Treatment, which allows most patients to avoid amputation, significantly improves the quality of life, improves the prognosis, and has a significant improvement in survival. However, the high invasion and metastasis ability of osteosarcoma and the resistance to chemotherapeutic drugs make some patients still have early recurrence or early metastasis.P27 after receiving standardized surgery and chemotherapy. A inhibitory factor in the cell cycle G1/S stage plays a key role in maintaining a smooth cell cycle. In vitro studies have found that, as a cell cycle inhibitor, p27 can not only inhibit the proliferation and migration of normal vascular endothelial cells, but also induce apoptosis. It is found that p27 can inhibit the primary swelling in the body. The growth of tumor and metastatic tumor, the deletion of p27 gene is one of the important reasons for the occurrence and progression of osteosarcoma. According to the report, the p27 gene in the nude mice model of human osteosarcoma transplanted tumor can improve the prognosis, relieve symptoms and prolong the survival time of tumor nude mice. The effect of soft hard and loose knot can promote blood circulation, eliminate blood stasis, use the treatment of digestive system tumor and prevent the liver damage in the process of tumor chemotherapy. The pathological model of osteosarcoma can be formed in the bone marrow cavity of nude mice. Adeno-related virus can be used as a carrier of p27 gene and expression of p27 protein in animals, and it has related biological activity. Therefore, this experiment selected human osteosarcoma Saos-2 cell lines, established the orthotopic xenograft model in nude mice, and used adeno-related virus as the p27 gene carrier. The inhibitory effects of p27 gene, Chinese herbal medicine and the combination of them on the growth of human osteosarcoma were observed and compared, and the experimental basis was provided for the gene therapy of osteosarcoma and the treatment of traditional Chinese medicine.
Experimental materials
1, cells, drugs and reagents: human osteosarcoma Saos-2 cell line, purchased in Shanghai Chao Yan Biological Technology Co., Ltd. (mainly composed of musk, bezoar, 37, snake gall), purchased in Zhangzhou Zai Zai Jin Pharmaceutical Co., Ltd. (production batch number: H.M.L.N.Z20080011); recombinant adeno-associated virus solution transfected with raav-p27, unloaded body weight group Adeno-related virus (rAAV-MCS) suspension, purchased in Xi'an Huaguang bioengineering company; fetal bovine serum (FBS), purchased in HyClone (NE, USA), p27 antibody, purchased in Shanghai Life Science Development Co., Ltd., enzyme chain avidin biotin (SABC) kit, purchased in Shanghai compound Biotechnology Co., Ltd., Rabbit anti beta -actin antibody, American Santa Cruze company products, horseradish peroxidase labelled Sheep anti rabbit two resistance, American Santa Cruze company products; ECL kit, Santa Cruze company products in the United States; BCA protein concentration test kit, American Bio-Rad company product.
2, the main instrument and equipment: blood red cell counter (Shanghai Laboratory Instrument Co., Ltd., China); semi dry transfer printer (Semidry Transfer system, American Bio-Rad company); ultraviolet gel automatic imaging instrument (Shanghai Qin Xiang Science Instrument Co., Ltd., China)
Experimental method
1, experimental animals:
BALB/C nude mice 30, SPF class no special pathogens, 3-4 weeks old, weight 15-20g, male and female, the specification is: CAnN.Cg-Foxn1nu/Cr1VR, purchased from Shanghai Bao Life Science Development Co., Ltd.. Feeding conditions: constant temperature 25-27, humidity 45%-50%, half barrier system environment, food, water, efficient filtration system treated The air is ventilated 10-15 times per hour, 10 hours a day, and 14 hours under dark conditions without lighting.
2, the cell culture of human osteosarcoma Saos-2 cell line:
Preparation of cell culture solution: using Roswell Park Memorial Institute (RPMI) -1640 culture solution, adding 10% standard fetal bovine serum, 2mmol/L- glutamyl ammonium, 100U/ml penicillin and 0.1mg/ml streptomycin, after filtration and disinfection, stored at 4 C. The cryopreservation tube containing human osteosarcoma Saos-2 cell line is removed, thawing, washing, and away from the liquid nitrogen tank. After the heart treatment, the cell suspension was transferred to the 10ml culture dish and placed in the 5%CO2 culture box for the night. When the cells grew to the relative density of about 80%, the cells were added to the digestive juice, digested at 37, 5% CO2 incubator for 2-3 minutes, and then added to the 2ml cell culture to terminate the cell digestion. At this time, the majority of the cells could be observed under the microscope. Free round cells. Stir the cells thoroughly with a straw and set aside. Discard the supernatant by centrifugation, add 5 ml cell culture medium and blow evenly. Count the cells with a cell counter and reserve them.
3, under aseptic conditions, the nude mice were anesthetized with 4% chloral chloral (10 L/g). Then the 0.1ml cell suspension (1 x 107 cells / syringes) was injected into the right armpit of the nude mice with the 1ml specification syringe needle 4 needle. After each injection of two nude mice for.2-4 weeks, it was observed that the subcutaneous transplanted tumor of the nude mice was grown to 1.0cm * 1.0cm x 1.5cm, and was executed. In nude mice, the osteosarcoma tissue was removed under the aseptic condition. After the axillary tumor tissue was treated as 0.1cm x 0.1cm x 0.1cm, the bone marrow cavity of the right hind leg of the nude mice was retransplanted into the bone marrow cavity of the right hind leg of the nude mice. Then the nude mice were put back in the cage to observe the growth of the tumor. At this time, the human osteosarcoma Saos-2 cell line in nude mice was established successfully. After inoculation of the tumor cells, the animals grew well. After 5 days of inoculation of the Saos-2 cell line, the tumor tissue had been growing subcutaneously in the nude mice. After six weeks of inoculation, no animal death was found, and the relative volume growth rate of the tumor was 92.7%.
4, experimental grouping and processing:
Thirty nude mice with similar tumor volume were randomly divided into five groups at the time of tumor growth of 0.8 cm after one week of inoculation.
In blank control group, tumor sites were injected with 100 micron LPBS for 1 days in 3 days.
In empty vector control group, the tumor site was injected with 100 mu L polyclonal site recombinant adeno-associated virus (rAAV-MCS) suspension. The concentration of empty vector virus solution was 1 *1011/L, once every 3 days.
After the tumor was formed, the drug group was given 0.01ml/g dose once a day, two times a day.
The p27 genome was injected with 100 L raav-p27 virus solution. The concentration of virus solution was 1 x 1011/L injection site, 3 days 1 times.
5. In the combined treatment group, the tumor site was injected with 100 mu L raav-p27 virus solution once every 3 days, and the tablets were fed with gall solution twice a day.
After inoculation in the nude mice, the mental state, weight, appetite, feces and urine were observed and recorded. The death of nude mice was found in the experiment and the cause of death was dissected and analyzed.
After 6 weeks of inoculation of tumor cells, all nude mice were killed by dislocated cervical vertebra. The tumor tissue was stripped, weighed and recorded, and the tumor growth inhibition rate was recorded. The formula was as follows: (control group tumor weight experimental group tumor weight) / control group of tumor weight x 100%. was established by using vernier caliper to measure the 1,4,8,12,16 of nude mice in each group. On the 18th and 22nd days, the maximum and minimum diameters of the tumor were calculated by Steel's formula V=1/2ab2, where a and B were the maximum and minimum diameters of the tumor, respectively.
5, immunohistochemical staining: the tumor tissue was immobilized with formalin, and the paraffin was embedded in the immunohistochemical staining method (IHC). The IgG (two anti) of the horse anti rat rat was labeled with anti p27 antibody (one antibody) and alkaline phosphatase, and the streptomycin biotin complex (SABC method) was used. The protein expression of p27 was detected by double staining. The expression of p27 was measured by optical microscopy, and the expression of positive cells was counted in a 10 fold field of view.
6, Western blot analysis: extract protein from tumor tissue cells, and then use anti p27 rabbit monoclonal antibody and Rabbit anti beta -actin internal reference antibody to carry out immunoblotting test (Western-Blot). After exposure to ECL chemiluminescence substrate, the protein content was calculated by laboratory image analysis software.
7, statistical analysis: the SPSS10.0 analysis software program was used to carry out statistical analysis. The data used mean mean standard deviation (X + S), and the one-way ANOVA and LSD analysis were carried out with the contrast group.
Result
1, the observation of the animal model: after the tumor cells were transplanted into the nude mice for 7 weeks, the p27 genome, the combined treatment group and all the nude mice were alive, with 1 deaths in the blank control group and the empty carrier control group. The growth of the tumor volume in the blank control group and the empty carrier group was very obvious, and two There was no significant difference in the mental state and appetite of the nude mice, the dark color of the urine, the dry feces and the loss of weight. The tumor growth of the nude mice was relatively slow, the mental state was good, the appetite was normal, the body weight changed little, the color of the feces and urine and the characters of the nude mice in the normal.P27 genome were the same. Slow, general mental state, a slight decline in appetite, weight increase with the increase of tumor volume, but the body is thin and occasional diarrhea. The tumor growth inhibition in the combined treatment group is obvious, the mental state of the nude mice is very good, the diet, feces and urine are normal, there is no diarrhea phenomenon, but the weight has a slight decline.
2, the tumor growth and inhibition rate: the volume of tumor in the blank control group and the empty vector control group was similar (P0.05). In the comparison of the tumor bodies recorded on the 8,12,16,18 and the 22 days, the p27 genome and the tablet group were significantly lower than those in the blank control group (P0.05). In comparison with the average weight of the tumor (P0.05), the drug group and the p27 genome were lighter than those in the blank control group (P0.05). The combined treatment group was lighter than that of the drug group or the p27 genome (P0.05). Therefore, in the tumor inhibition rate, the drug group and the p27 genome were higher than the blank control group (P0.05). The combined treatment group was also higher than that of pizin medicine group or p27 genome (P0.05).
3, p27 protein expression: after dyed p27 protein positive cells, the brown yellow granules can be observed in the nucleus. In the positive rate of p27 protein, the p27 genome is significantly higher than that in the blank control group (P0.05). The combined treatment group is also significantly higher than the p27 genome or the tablet group (P0.05). The Western blot test shows that in P2, the immunoblotting test shows P2 Compared with the blank control group, p27 genome was higher (P 0.05), and the combined treatment group was higher than p27 genome or litter gall (P 0.05).
conclusion
1, Saos-2 human osteosarcoma cell line can be transplanted into nude mice to obtain an animal model of tumor growth in situ.
2. Adeno-associated virus as a gene vector has no effect on tumor growth. Adeno-associated virus transfected with p27 gene can increase the expression of p27 protein in tumor tissue.
3. Both p27 gene and Tablet gall can inhibit the growth of osteosarcoma, and the inhibitory effect on the growth of osteosarcoma is obviously enhanced when they are combined.
Significance
The recombinant adeno-associated virus (p27) can be used in the study and treatment of osteosarcoma, and the combination of p27 gene and traditional Chinese medicine has synergistic effect on the inhibition of osteosarcoma. The possibility of adeno-related virus as a gene carrier for tumor gene therapy is preliminarily discussed. In the treatment of osteosarcoma, the p27 gene is combined with Chinese herbal medicine. The research of curative effect has laid the theoretical foundation further.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R738.1
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