小鼠胚胎干细胞与黑色素瘤B16细胞体外相互作用的初步研究

发布时间：2018-04-15 01:39

本文选题：饲养层 + 胚胎干细胞　；参考：《重庆医科大学》2012年硕士论文

【摘要】：恶性肿瘤是增殖失控和异常分化的细胞集合体，其生物学行为与早期胚胎发育的生命过程极为相似。转移性癌细胞类似于干细胞，它们同时表现自我更新和相似的细胞分子标志，显示两种细胞类型都有一个基本的可塑性。如果将两种极为类似的细胞放在一起培养会产生怎样的现象呢？谁会占优势，或者谁会更多的对另一方产生影响以及会产生怎样的影响呢？本研究拟建立C57BL／6小鼠胚胎干细胞系，着眼于将小鼠胚胎干细胞与小鼠恶性黑色素瘤B16细胞进行共培养，探索共培养中两种细胞相互作用情况，以及胚胎干细胞与肿瘤细胞共培养对肿瘤细胞生物学行为的影响。目的 1.优化小鼠胚胎成纤维细胞饲养层制备条件，以建立稳定高效的C57BL／6小鼠胚胎成纤维细胞饲养层培养体系，用于小鼠胚胎干细胞的纯化和建系培养。 2.建立稳定的C57BL／6小鼠胚胎干细胞系。 3.通过小鼠胚胎干细胞与B16细胞体外共培养，及检测共培养后B16细胞部分生物学行为变化，初步探讨小鼠胚胎干细胞与B16细胞体外相互作用及其机制。方法 1.取妊娠12.5-14.5d的胎鼠，组织消化法分离培养MEF；MTT法检测MEF细胞增殖活性，筛选出制备饲养层中合适的丝裂霉素C作用MEF细胞的浓度时间、细胞接种密度及细胞代数。 2.妊娠3.5d的小鼠囊胚置于不同接种密度饲养层细胞培养体系上，观察囊胚、内细胞团及ES细胞的生长状况，，进一步验证优化后MEF饲养层分离培养ES细胞的效果。 3.C57BL／6小鼠3.5d的囊胚置于小鼠胚胎成纤维细胞制作的饲养层上孵育，3-5d后分离内细胞团，扩增传至35代以上，观察集落生长情况，通过形态学、碱性磷酸酶染色、早期胚胎特异性抗原（SSEA-1）和OCT-4染色，及体内分化实验对ES细胞集落进行鉴定。 4.肿瘤细胞先接种后加入胚胎干细胞，胚胎干细胞先接种后加入肿瘤细胞，以及两种细胞同时接种，采用ES条件培养基进行共培养；及肿瘤细胞先接种后加入胚胎干细胞，采用DMEM／F12+20%FBS培养基进行共培养，筛选合适的小鼠胚胎干细胞与B16细胞共培养体系。 5.通过小鼠胚胎干细胞与B16细胞体外共培养模型观察小鼠胚胎干细胞与B16细胞共培养中相互作用，MTT法检测共培养后B16细胞增殖能力及粘附能力的变化，transwell小室法检测共培养后B16细胞侵袭及迁移能力的变化。结果 1.丝裂霉素C10ug／ml作用3-3.5h、20ug／ml作用2-2.5h抑制MEF细胞效果较好；MEF以（1-2）×104／孔接种于96孔板，比以（3-6）×104／孔接种的细胞活力稳定；丝裂酶素C处理MEF后1-4代比5、6代活力稳定，活力维持时间较长。 2.饲养层细胞不同接种密度培养体系下分析囊胚贴壁率、内细胞团孵出率及ES细胞克隆形成率，进一步验证结果1中筛选出来的条件下制备的饲养层细胞囊胚贴壁率、内细胞团孵出率及ES细胞集落克隆形成率均较其他条件下高，最终可以分离培养出可稳定传代的ES细胞集落。 3.分离培养后得到的ES细胞可稳定传代至35代以上，且均呈集落样生长，AKP染色、免疫组化SSEA-1表面抗原和OCT-4抗原均为阳性，接种于裸鼠及C57BL／6小鼠均可形成畸胎瘤，HE染色后均有三个胚层组织成分，符合小鼠ES细胞的特性。 4.四种不同条件下小鼠胚胎干细胞与B16细胞共培养，建立了合适的小鼠胚胎干细胞与B16细胞共培养体系。 5.小鼠胚胎干细胞与B16细胞共培养中小鼠胚胎干细胞能够侵入并推开小鼠黑色素瘤B16细胞形成自己的生长空间；与对照组比较共培养后肿瘤细胞的增殖能力、粘附能力、侵袭及迁移能力均显著降低（P＜0.05，P＜0.01)。结论优化条件后制备的小鼠胚胎成纤维细胞饲养层可以较好地支持囊胚贴壁、ICM孵出及ES细胞集落形成与生长，获得的ES细胞经鉴定符合小鼠ES细胞的一般特性，稳定传至35代以上仍保持良好的胚胎干细胞干性特征，建立了稳定的C57BL／6小鼠胚胎干细胞系。利用共培养实验模型，进行小鼠胚胎干细胞与B16细胞体外共培养，体外共培养中小鼠胚胎干细胞能够侵袭肿瘤细胞并形成自己的生长空间，并使B16细胞体外增殖能力、粘附能力、侵袭及迁移能力降低。
[Abstract]:Malignant tumor cell aggregates proliferation and abnormal differentiation and its biological behavior and early embryonic development process of life are very similar. Metastatic cancer cells similar to stem cells. They also showed similar self-renewal and cell molecular markers, showed that two kinds of cell types have a basic plasticity.
If two is very similar to the cell culture together will produce what kind of phenomenon? Who will be dominant, or who would have more influence on the other side, what are the effects? This study intends to establish C57BL / 6 mouse embryonic stem cell lines, focusing on the mouse embryonic stem cells with the mouse melanoma B16 cells were co cultured, to explore the co culture two cell interactions, and embryonic stem cells co cultured with tumor cells on the biological behavior of tumor cells.
objective
1., we optimized the preparation conditions of feeder layer of mouse embryonic fibroblast, so as to establish a stable and efficient C57BL / 6 mouse embryonic fibroblast feeder layer culture system, which was used for purification and establishment of mouse embryonic stem cell culture.
2. a stable C57BL / 6 mouse embryonic stem cell line was established.
3., in vitro co culture of mouse embryonic stem cells and B16 cells, and detection of some biological behavior changes of B16 cells after co culture, and preliminarily explore the interaction between mouse embryonic stem cells and B16 cells in vitro and its mechanism.
Method
1. fetch mice with gestational 12.5-14.5d, MEF was isolated by tissue digestion, MTT assay was used to detect the proliferation activity of MEF cells, and the appropriate mitomycin C level, MEF cell density, cell density and cell algebra were screened out.
2. the 3.5D blastocyst of pregnant mice was placed on different feeder density cell culture system. The growth status of blastocyst, inner cell mass and ES cell were observed, and further verified the effect of MEF feeder layer separating and culturing ES cells.
The fibroblast feeder layer were incubated with mouse embryos at the blastocyst in 3.C57BL / 6 mice 3.5D, isolated inner cell mass after 3-5d was passaged over 35 times to observe the colony growth, the morphology, alkaline phosphatase staining, early embryonic antigen (SSEA-1) and OCT-4 staining, and in vivo experiment on differentiation ES cells were set to identify.
Embryonic stem cells with 4. tumor cells before inoculation, embryonic stem cells first after inoculation with the tumor cells, and two cell inoculation, were co cultured with ES medium conditions; embryonic stem cells into tumor cells and first after inoculation with DMEM / F12+20%FBS co culture medium, screening of the co culture system of mouse embryo the appropriate stem cells and B16 cells.
The 5. mouse embryonic stem cells in vitro and B16 cell model of mice embryonic stem cells co cultured with B16 cells in co culture interaction, change MTT assay after co cultured B16 cells proliferation and adhesion ability, change of Transwell assay was co cultured with B16 cells after the invasion and migration ability.
Result
1. C10ug / ml mitomycin 3-3.5h, 20ug / ml 2-2.5h MEF cell inhibitory effect is better; MEF (1-2) * 104 / inoculated in 96 pore plate, compared with (3-6) * 104 / stable cell viability inoculated; mitomycin C treatment after MEF 1-4 5,6 than the generation of stable activity activity to maintain a longer time.
Blastocyst attachment rate analysis of 2. feeder cell culture system under different inoculation density, hatching rate of inner cell mass and ES cell clone formation rate, further verify the results of screening out 1 conditions in the preparation of feeder cells of blastocyst attachment rate, hatching rate of inner cell mass and ES cell colony formation rate under other conditions, can be isolated and cultured stably passaged ES cell colony.
3. isolation of ES from the cells was stable for more than 35 passages, and showed colony like growth, AKP staining, immunohistochemistry of SSEA-1 surface antigen and OCT-4 antigen were positive in nude mice and C57BL / 6 mice can form teratomas, HE staining were three embryonic tissue components, in line with the characteristics of mouse ES cells.
4. the mouse embryonic stem cells were co cultured with B16 cells under four different conditions, and a suitable co culture system of mouse embryonic stem cells and B16 cells was established.
Mouse embryonic stem cells can invade and open the B16 mouse melanoma cells formed their own growth space co cultured 5. mouse embryonic stem cells and B16 cells; compared with the control group were co cultured with tumor cells after proliferation, adhesion ability, invasion and migration were significantly lower (P < 0.05, P < 0.01).
conclusion
The optimum conditions for the preparation of mouse embryonic fibroblast feeder layer can effectively support blastocyst adherence, ICM hatch and ES cell colony formation and growth, the ES cells were identified in line with the general characteristics of mouse ES cells, stably passaged more than 35 passages of embryonic stem cells still maintain a good dry characteristic, established C57BL 6 / mouse embryonic stem cell lines. The stable experimental model of co culture, co culture of mouse embryonic stem cells and B16 cells in vitro, and the mouse embryonic stem cells to invasion of tumor cells and the formation of their growth space were cultured in vitro, and the proliferation ability of B16 cells in vitro adhesion, reduce the invasion and migration.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

【参考文献】