LB-100对宫颈癌细胞Ca ski辐射敏感性及肠道作用的实验研究

发布时间：2018-07-31 16:44

【摘要】：背景和目的社会人口老龄化及环境污染,致使恶性肿瘤的发病率不断升高,成为导致死亡的最主要原因。在妇科肿瘤中,宫颈癌的死亡率最高,且近年来出现了年轻化的趋势。尤其是在中国等亚洲国家,HPV病毒广泛感染,导致宫颈癌在这些国家的发病率更高。早期宫颈癌的治疗手段主要是放疗,或手术联合放疗,效果较好。但是,放射线对组织的破坏作用是无选择性的,且宫颈位于盆腔,其邻近组织——肠道,对放射线异常敏感。因此,宫颈癌接受放疗的患者常不可避免地并发放射性肠损伤,患者表现为腹痛、腹泻、便血,远期甚至会导致肠梗阻等严重情况,加重了患者心理、生理以及经济上的负担,也极大地加重了临床护理的工作量。若通过降低放疗剂量来避免肠道并发症,又会导致宫颈癌消除不彻底,远期发生转移和复发。因此,需要一种能增强宫颈癌细胞辐射敏感性的药物,联合放疗来治疗宫颈癌。以此在不影响抗肿瘤效果的情况下,可降低辐射剂量,尽可能地避免放射性肠损伤发生,进而可减轻患者及临床护理工作的负担。PP2A(Protein phosphatase 2,蛋白磷酸酶)是一种参与细胞周期、DNA损伤应答等的调节蛋白。研究证明,外源性抑制剂,如岗田酸和斑螯素,通过抑制PP2A的活性,改变肿瘤细胞的细胞周期进程及DNA损伤应答机制,从而增强肿瘤细胞对放化疗的敏感性。LB-100是PP2A的一种新研发的水溶性抑制剂,动物荷瘤模型上证明其能够有效增强骨肉瘤、卵巢癌等移植瘤对放化疗的敏感性,且其对肝、肾等重要组织脏器无明显毒副作用,目前处于Ⅰ期临床研究阶段。但还未见LB-100对宫颈癌作用的相关研究报道。由于宫颈癌放疗常不可避免地造成放射性肠损伤,患者承受着生理和心理上的痛苦,也促使其治疗和护理面临巨大的困难,因此在研究增强宫颈癌放疗敏感性药物的同时,应该关注该药物对肠道组织的作用。综合以上因素,本研究以Ca ski(人宫颈癌细胞株)和CCD 841 Co N(人正常肠上皮细胞株)及HIEC(人正常肠上皮细胞株)为研究对象,探讨了PP2A抑制剂LB-100对宫颈癌细胞株Ca ski辐射敏感性的影响,以及其对正常肠上皮细胞株CCD 841 Co N及HIEC增殖及辐射敏感性的影响。同时,本研究利用小鼠放射性肠损伤模型,初步观察了LB-100对小鼠正常肠道组织以及放射性肠损伤的肠道组织的影响,以期为宫颈癌的临床放射治疗和患者受照后的护理提供一定的指导。研究方法:1.LB-100药物浓度筛选:梯度浓度的LB-100分别处理Ca ski、CCD 841 CoN和HIEC细胞,24hr后用CCK-8法检测以上细胞的细胞活性,最终选择对两株人正常肠上皮细胞无抑制作用的浓度作为安全给药浓度。2.48hr细胞活性检测(Ca ski、CCD 841 Co N和HIEC细胞):将细胞分别分组为对照组(control)、单纯LB-100组(LB)、单纯辐照组(R)、LB-100联合辐照组(LB+R),给予低浓度LB-100(2.5μM)和(或)辐照(6Gy)处理,辐照(6Gy)后48hr,以CCK-8试剂检测细胞活性。3.平板克隆形成实验(Ca ski、CCD 841 Co N和HIEC细胞):细胞分组方法同2,分别给予LB-100(2.5μM)和(或)辐照(0Gy、2Gy、4Gy、6Gy)处理,24hr后去除LB-100继续培养,至克隆形成后进行染色并计数。4.流式细胞术凋亡检测(Ca ski):细胞分组方法同2,LB-100(2.5μM)和(或)辐照(6Gy)处理人宫颈癌细胞株Ca ski,3hr后给予辐照(6Gy),于辐照后24hr和48hr以流式细胞术检测凋亡。5.流式细胞术细胞周期检测(Ca ski):细胞分组和处理方法同4,于辐照后24hr以流式细胞术检测细胞周期。6.免疫荧光染色(Ca ski):细胞分组为对照组(control)和单纯LB-100组(LB),LB-100(2.5μM)处理48hr后,进行免疫荧光染色(DAPI和Tublin)。7.体内实验:BALB/c小鼠分组为对照组(control)、单纯LB-100组(LB)、单纯辐照组(R)、LB-100联合辐照组(LB+R)。给予LB-100(2.5mg/kg)或等体积生理盐水腹腔注射,3hr后给予一次性全身辐照11Gy或0Gy。并于辐照后24hr、48hr、72hr分别给予一次LB-100(2.5mg/kg)或者等体积生理盐水腹腔注射并观察记录小鼠基本状态,于辐照后84h颈部脱臼处死小鼠,获取小鼠肠道组织,测量小肠肠度,进行肠道组织HE和Brdu染色。结果:1.LB-100药物浓度筛选结果显示:当浓度小于5μM时,LB-100对以上三种细胞的细胞活性均无明显抑制作用,当浓度≥5μM时,LB-100对以上三种细胞活性的抑制作用明显,呈浓度依赖趋势。2.48hr细胞活性检测结果显示:与control组比较,Ca ski细胞的LB组细胞活性降低,而CCD 841 Co N和HIEC的LB组细胞活性无差异,以上三种细胞的R组的细胞活性均降低。与R组比较,Ca ski的LB+R组的细胞活性降低更明显,而CCD 841Co N和HIEC的LB+R组细胞活性与R组比较无显著差异。3.平板克隆形成实验结果显示:Ca ski和CCD 841 CoN的LB组的克隆数小于control组,而HIEC的LB组的克隆数与control组比较无差异。以上三种细胞的R组的细胞克隆数均小于control组。Ca ski的LB+R组的细胞克隆数少于R组和LB组。CD 841 CoN和HIEC的LB+R组的克隆数与R组比较无显著差异。4.Ca ski细胞凋亡凋亡结果显示:LB组和R组凋亡率显著高于control组,LB+R组的凋亡率明显高于单纯LB和R组。5.Ca ski细胞周期检测结果显示:LB组和R组细胞周期的G0/G1期比例低于control组,但LB+R组G0/G1期比例低于LB组和R组。6.Ca ski细胞免疫荧光染色结果显示:control组Ca ski细胞核形态正常及微管结构完整,而LB组大量异常核出现且细胞核体积较control组大,微管的结构消失。7.从小鼠的体重等基本情况、肠道组织的病理切片HE染色以及隐窝增殖染色的情况来看,LB组与control组比较无显著差异。LB+R组与R组小鼠的体重下降,小肠长度变短,肠上皮结构被严重破坏,但两组之间比较无差异。结论:1.浓度低于5μM时,LB-100对Ca ski细胞无明显毒性作用,可选择较低浓度LB-100(2.5μM)作为后续实验的给药浓度。低浓度LB-100能增强宫颈癌细胞Ca ski的辐射敏感性。LB-100增强Ca ski细胞辐射敏感性的可能机制是诱导Ca ski细胞凋亡,促进Ca ski细胞的细胞周期进程,破坏Ca ski细胞微管结构,导致多倍体及异常核形成,最终导致Ca ski细胞发生有丝分裂性死亡。2.浓度低于5μM时,LB-100对CCD 841 Co N和HIEC细胞无明显毒性作用,视作安全浓度范围。低浓度LB-100对正常肠上皮细胞CCD 841 Co N和HIEC的辐射敏感作用无影响。LB-100对小鼠正常肠道组织无副作用,且未加重放射性肠损伤模型小鼠的肠损伤程度,但其对损伤的肠道组织也无明显保护作用。
[Abstract]:Background and objective social population aging and environmental pollution, resulting in the increasing incidence of malignant tumor, which has become the main cause of death. In gynecologic tumors, the mortality of cervical cancer is the highest, and in recent years, the trend of youth has emerged. Especially in Asian countries such as China, HPV virus is widely infected, causing cervical cancer in these The incidence of the country is higher. The treatment of early cervical cancer is mainly radiotherapy, or surgery combined with radiotherapy, the effect is better. However, the destruction of the tissue is not selective, and the cervix is located in the pelvic cavity, its adjacent tissue - intestinal, sensitive to radiation. For this reason, the patients receiving radiotherapy of cervical cancer are often unavoidable and Radiative intestinal injury, with abdominal pain, diarrhea, blood pressure, long term and even cause severe intestinal obstruction, aggravates the psychological, physiological and economic burden of the patient, and it also greatly aggravates the workload of clinical nursing. If the radiation dose is reduced to avoid intestinal complications, it will lead to the elimination of cervical cancer and the long term. There is a need for a drug that can enhance the radiosensitivity of cervical cancer cells and combined with radiotherapy to treat cervical cancer. In this case, the radiation dose can be reduced without affecting the antitumor effect, and the radiation intestinal damage can be avoided as far as possible, and the burden of light patients and clinical nursing work can be reduced by.PP2A (Protein pH). Osphatase 2, protein phosphatase) is a regulatory protein that participates in cell cycle, DNA damage response and so on. Studies have shown that exogenous inhibitors, such as Pantian acid and chelatosin, change the cell cycle process of tumor cells and the mechanism of DNA damage response by inhibiting the activity of PP2A, thus enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy.LB-100 is PP2A A newly developed water soluble inhibitor, animal bearing tumor model proves that it can effectively enhance the sensitivity of osteosarcoma and ovarian cancer to radiotherapy and chemotherapy, and it has no obvious toxic and side effects on important organs such as liver and kidney. It is currently in phase I clinical study stage, but there is no Related Research Report on the effect of LB-100 on cervical cancer. As cervical cancer is often caused by radiation intestinal damage, patients suffer from physiological and psychological pain and are also facing great difficulties in the treatment and nursing. Therefore, we should pay attention to the role of the drug on the intestinal tissue while strengthening the radiotherapy sensitive drugs for cervical cancer. This study is based on the above factors. This study is based on Ca sk I (human cervical cancer cell line) and CCD 841 Co N (human normal intestinal epithelial cell strain) and HIEC (human normal intestinal epithelial cell strain) were studied. The effect of PP2A inhibitor LB-100 on the ski radiosensitivity of cervical cancer cell line Ca, and its effect on CCD 841 Co N, proliferation and radiosensitivity of normal intestinal epithelial cell line, were also discussed. The effect of LB-100 on normal intestinal tissue and intestinal tissue injury in mice was preliminarily studied by using the model of radiation intestinal injury in mice. In order to provide some guidance for the clinical radiation therapy of cervical cancer and the nursing care of the patients after exposure, the method of screening the concentration of 1.LB-100: the gradient concentration of LB-100 respectively Ca ski, CCD 841 CoN and HIEC cells were used to detect the cell activity of the above cells by CCK-8 method after 24hr. Finally, the concentration of the two normal intestinal epithelial cells was selected as a safe dose of.2.48hr cell activity (Ca ski, CCD 841 Co). LB), simple irradiation group (R), LB-100 combined irradiation group (LB+R), low concentration LB-100 (2.5 u M) and (or) irradiation (6Gy) treatment, and 48hr after irradiation (6Gy), and CCK-8 reagent to detect cell activity.3. flat plate clone formation experiment. After 24hr, LB-100 was removed and continued to be cultured, stained after cloning, and counted.4. flow cytometry (Ca ski). Cell grouping method was treated with 2, LB-100 (2.5 M) and / or irradiation (6Gy) to treat the Ca ski of human cervical cancer cell line, and irradiated (6Gy) after 3hr, and the apoptotic cells were detected by flow cytometry after irradiation. Cell cycle detection (Ca ski): cell grouping and treatment were 4, and cell cycle.6. immunofluorescence staining (Ca ski) was detected by flow cytometry in 24hr after irradiation. Cells were grouped into control group (control) and LB-100 group (LB), LB-100 (2.5 mu M) were treated for 48hr, and immunofluorescence staining in vivo was carried out. Group as control group (control), simple LB-100 group (LB), simple irradiation group (R), LB-100 combined irradiation group (LB+R). Give LB-100 (2.5mg/kg) or equal volume of physiological saline intraperitoneal injection, 3hr after 3hr and irradiate 11Gy or 0Gy. and irradiated 24hr. The basic state of the mice was observed and the mice were dislocated in the 84h neck after irradiation. The intestinal tissue of the mice was obtained, the intestinal intestinal degree was measured and the intestinal tissue was stained with HE and Brdu. The results showed that the concentration screening results of 1.LB-100 showed that when the concentration was less than 5 mu M, LB-100 had no obvious inhibitory effect on the cell activity of the above three cells. The inhibitory effect of LB-100 on the activity of the above three cells was obvious, and the activity of.2.48hr cells in the concentration dependent trend.2.48hr cell activity showed that the activity of LB group in Ca ski cells decreased and the activity of CCD 841 Co N and HIEC LB group was no difference compared with that of the control group. The cell activity of Ca ski LB+R group decreased more obviously, while the cell activity of LB+R group of CCD 841Co N and HIEC had no significant difference compared with the R group. The experimental results of.3. plate clone formation showed that the number of clones in Ca ski and 841 groups was less than that of the group, and the number of clones in the group was not different from that of the group. The number of cell clones was less than that of group control.Ca ski, the number of cell clones in group LB+R was less than that of R group and LB group.CD 841 CoN and HIEC LB+R group, there was no significant difference between R group and R group. The results of cell cycle detection showed that the G0/G1 phase ratio of cell cycle in group LB and R group was lower than that in group control, but the proportion of G0/G1 phase in LB+R group was lower than that of LB group and R group.6.Ca ski cell immunofluorescence staining results showed that control group was normal and the structure of microtubule was complete, while large amount of abnormal nuclei appeared and nucleus volume was larger than that of group LB+R. The structure of microtubule disappeared.7. from the basic condition of the weight of mice, the pathological section of the intestinal tissue, HE staining and the coloring of the recess, there was no significant difference between the group LB and the control group, and the weight of the mice in the.LB+R group and the R group decreased, the length of the small intestine was shorter, and the intestinal epithelium was seriously damaged, but there was no difference between the two groups. Conclusion: 1 When the concentration is less than 5 M, LB-100 has no obvious toxic effect on Ca ski cells, and the lower concentration LB-100 (2.5 u M) is selected as the dosage of the following experiment. The low concentration LB-100 can enhance the radiosensitivity of Ca ski in cervical cancer cells, and the possible mechanism of enhancing the radiosensitivity of Ca ski cells is to induce apoptosis and promote the cell apoptosis. The cell cycle process disrupts the microtubule structure of Ca ski cells and causes polyploidy and abnormal nucleus formation, which eventually leads to the mitotic death of Ca ski cells and the.2. concentration is lower than 5 u M, and LB-100 has no obvious toxic effect on CCD 841 Co N and HIEC cells, which is considered as a safe concentration range. Low concentration LB-100 is 841 of normal intestinal epithelial cells. The radiation sensitivity of C did not affect the side effects of.LB-100 on normal intestinal tissue in mice, and did not aggravate the degree of intestinal damage in mice with radiation intestinal damage, but it had no significant protective effect on the injured intestinal tissue.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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