黑色素瘤小鼠高转移模型体内筛选和细胞系的建立

发布时间：2018-06-06 23:32

本文选题：黑色素瘤 + 转移　；参考：《扬州大学》2008年硕士论文

【摘要】： 目的:采用深度免疫缺陷动物,并通过手术切除肿瘤进行高转移模型体内综合筛选的实验思路,建立鼠源和人源黑色素瘤小鼠高转移模型及相应细胞系,同时观察相关生物学特性,探讨转移相关机理,为肿瘤的预防、诊断和治疗等提供理想的动物模型。方法:1.小鼠黑色素瘤高转移模型的体内筛选和细胞系的建立: 将小鼠黑色素瘤B16移植于T、B、NK细胞联合免疫缺陷的BNX小鼠皮下,首先进行皮下移植→手术切除→肺转移→皮下移植的连续三代体内筛选,再按照皮下移植→肺转移→皮下移植的方法继续进行三代筛选,建立移植性自发高转移模型,同时进行肿瘤的生长、转移情况和病理组织学的观察。采用组织块法对小鼠黑色素瘤高转移肿瘤组织进行原代培养及建立细胞系,观察肿瘤细胞的形态学、生长速度、体外侵袭能力、染色体形态分析、细胞周期变化、体内验证皮下移植的成瘤率和转移情况。 2.人黑色素瘤高转移模型的体内筛选及微转移动态检测: 利用人黑色素瘤A357细胞株进行SCID小鼠皮下移植实验时,发现6只动物中有1只发生明显肺转移,取肺转移灶移植至SCID小鼠皮下扩增。采用皮下移植→肺转移→皮下移植体内反复筛选的方法,建立皮下移植小鼠肺高转移动物模型(血路转移合并淋巴道转移)及相应的人黑色素瘤高转移细胞系,并进行相关生物学特性的初步研究。同时应用该细胞系建立BNX小鼠皮下移植瘤模型,通过Alu-PCR进行动态、定性的肺微转移检测。结果:1.小鼠黑色素瘤高转移模型的体内筛选和细胞系的建立: 通过体内综合筛选,成功建立了BNX小鼠B16黑色素瘤皮下移植肺高转移模型。该模型的皮下移植成瘤率可达100%,35d时肺转移率达到91.7%(11/12) ,转移程度达到+++。同时建成相应的高转移细胞系,将其命名为B16-sci,其倍增时间为46.07h,自然凋亡率显著低于B16细胞(P0.05),体外侵袭能力显著强于B16细胞(P0.05),染色体分析结果表明B16-sci细胞符合小鼠恶性肿瘤细胞染色体的特点。体内验证结果表明,B16-sci细胞移植于BNX小鼠皮下,已能稳定形成100%(6/6)肺转移,同时提高了该细胞在C57BL/6J小鼠皮下移植后的肺转移率。 2.人黑色素瘤SCI-375高转移模型的筛选及微转移动态检测: 经体内反复筛选,成功建立了皮下移植小鼠肺转移动物模型(血路转移合并淋巴道转移)。潜伏期从筛选初期的7-14天逐渐缩短至5天左右,荷瘤寿命从60-75天下降并稳定在45天左右。筛选传代的243只SCID小鼠,皮下100%成瘤,肺转移率随着筛选的进行,逐代提高,第6代起肺转移率保持在100%。建成了相应的高转移细胞系,并将其命名为SCI-375,其倍增时间为21.60h,体外侵袭能力强于未筛选的A375(P0.05),染色体分析结果显示该细胞具有人类恶性肿瘤细胞染色体的特点。体内验证结果表明:SCI-375在不同免疫缺陷动物体内均能表达较高的转移率, BNX小鼠肺转移率为100%(13/13),裸小鼠肺转移率为90.91%(10/11)。Alu-PCR结果显示:2W时,PCR检测和常规病理检测肺转移均为阴性;3W、4W、5W时,PCR检测肺转移率分别为(37.5%)3/8、(75%)6/8、(100%)10/10,常规病理检测肺转移率分别为(0%)0/8、(37.5%)3/8、(50%)5/10。可见,分子生物学的方法可以早期检测肿瘤微转移情况的发生,其灵敏度和高效性要优于常规病理检测。结论:1.利用深度免疫缺陷动物以及手术切除肿瘤进行高转移模型的体内筛选的实验思路,成功建立了一个肺转移率高、转移特性稳定、直观性好、操作简便的B16黑色素瘤皮下移植小鼠自发高转移模型及相应的细胞系。 2.成功建立了人黑色素瘤皮下移植小鼠自发高转移模型及相应的细胞系,而且存在血路和淋巴两个转移途径;在不同免疫缺陷小鼠体内表达和应用并获得证实。用PCR技术的高敏感性,通过扩增特异的基因片段,对其在小鼠体内的微转移进行动态检测。 3.为探讨肿瘤转移的生物学机制和抗转移治疗提供了理想的动物模型及相应的细胞系。
[Abstract]:Objective: to establish a high metastasis model and the corresponding cell lines in mice and human melanoma mice, and to explore the related biological characteristics and explore the mechanism of metastasis related to the prevention, diagnosis and treatment of tumor. An animal model you want.
Methods: in vivo screening and cell line establishment of 1. high metastatic melanoma models in mice:
The murine melanoma B16 was transplanted subcutaneously into the T, B and NK cells combined with the immune deficient BNX mice. First, subcutaneous transplantation, surgical resection, pulmonary metastasis and subcutaneous transplantation were screened in three successive generations. Then the three generation screening was continued according to subcutaneous transplantation, pulmonary metastasis and subcutaneous transplantation, and a spontaneous high transfer model was established. The tumor growth, metastasis and histopathological observation were carried out. Tissue block method was used to carry out primary culture and cell lines in mice with high metastatic tumor of melanoma. The morphology, growth speed, invasion ability, chromosome morphology and cell cycle change of the tumor cells were observed, and the tumor formation of subcutaneous transplantation was verified in vivo. Rate and transfer.
In vivo screening and micrometastasis dynamic detection of 2. human melanoma models with high metastasis:
When the human melanoma A357 cell line was used for subcutaneous transplantation of SCID mice, it was found that 1 of the 6 animals had obvious pulmonary metastasis, and the lung metastasis was transplanted to the SCID mice subcutaneously. The animal model of lung high metastasis (blood transfer) was established by subcutaneous transplantation, pulmonary metastasis and subcutaneous transplantation in vivo. A preliminary study on the related biological characteristics of the human melanoma high metastatic cell line and the corresponding human melanoma metastasis cell line was carried out. The cell line was used to establish the model of the subcutaneous transplantation tumor in BNX mice, and the dynamic and qualitative lung micrometastasis was detected by Alu-PCR.
Results: 1. in vivo screening and cell line establishment of mouse melanoma high metastatic model:
Through the comprehensive screening in vivo, the lung high metastasis model of subcutaneous transplantation of BNX mice with B16 melanoma was successfully established. The rate of subcutaneous transplantation of this model could reach 100%, the lung metastasis rate of 35d reached 91.7% (11/12), the degree of metastasis reached + + + + +. At the same time, the corresponding high transfer cell line was set up to be B16-sci, and its doubling time was 46.07h and natural apoptosis. The rate was significantly lower than that of B16 cells (P0.05), and the ability of invasion in vitro was significantly stronger than that of B16 cells (P0.05). The results of chromosome analysis showed that B16-sci cells were in line with the characteristics of the chromosomes of the malignant tumor cells of mice. The results showed that the transplantation of B16-sci cells to the subcutaneous of BNX mice could stabilize the formation of 100% (6/6) lung metastasis and increase the number of the cells in C57B. The lung metastasis rate of L/6J mice after subcutaneous transplantation.
Screening of SCI-375 high metastatic model of 2. human melanoma and dynamic detection of micrometastasis:
After repeated screening in the body, the animal model of lung metastases (hematogenous metastasis combined with lymphatic metastasis) was successfully established. The incubation period was shortened from 7-14 days to 5 days, and the life span of the tumor was reduced from 60-75 days to 45 days. 243 subcutaneous mice were screened, 100% subcutaneous tumors were subcutaneously, and the lung metastasis rate was selected with screening. The sixth generation of lung metastasis rate in the sixth generation maintained a corresponding high metastatic cell line and named it SCI-375, and its doubling time was 21.60h, and the invasion ability in vitro was stronger than that of unscreened A375 (P0.05). The results of chromosome analysis showed that the cell has the characteristics of the chromosome of human malignant tumor cells. The results showed that SCI-375 could express high metastasis rate in different immune deficient animals. The lung metastasis rate of BNX mice was 100% (13/13), and the lung metastasis rate of nude mice was 90.91% (10/11).Alu-PCR. The results showed that PCR detection and routine pathological examination were negative when 2W, 3W, 4W, 5W, PCR detection of lung metastasis rate was (37.5%) 3/8, (75%), (100%) 10/10, the rate of lung metastasis by routine pathological examination was (0%) 0/8, (37.5%) 3/8, (50%) 5/10., and the molecular biology method could detect the occurrence of tumor micrometastasis in early stage, and its sensitivity and efficiency were better than that of routine pathological examination.
Conclusion: 1. a high transfer rate, stable, intuitionistic and easy operation of B16 melanoma subcutaneous transplantation model and the corresponding cell lines were successfully established by using the experimental method of screening the high metastasis model of the advanced immunodeficiency animals and the surgical resection of the tumor.
2. the spontaneous high metastasis model and the corresponding cell line in the subcutaneous transplantation of human melanoma were successfully established, and there were two transfer pathways of blood and lymph, and the expression and application in different immune deficient mice were confirmed. The specific gene fragment was amplified by the Gao Min sensibility of the PCR technique, and the micrometastasis in mice was amplified by the amplification of the specific gene fragments. Dynamic testing.
3. it provides an ideal animal model and corresponding cell lines for exploring the biological mechanism of tumor metastasis and anti metastasis therapy.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R739.5;R-332

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