本文選題：飼養(yǎng)層 + 胚胎干細(xì)胞�。� 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：惡性腫瘤是增殖失控和異常分化的細(xì)胞集合體，其生物學(xué)行為與早期胚胎發(fā)育的生命過(guò)程極為相似。轉(zhuǎn)移性癌細(xì)胞類似于干細(xì)胞，它們同時(shí)表現(xiàn)自我更新和相似的細(xì)胞分子標(biāo)志，顯示兩種細(xì)胞類型都有一個(gè)基本的可塑性。 如果將兩種極為類似的細(xì)胞放在一起培養(yǎng)會(huì)產(chǎn)生怎樣的現(xiàn)象呢？誰(shuí)會(huì)占優(yōu)勢(shì)，或者誰(shuí)會(huì)更多的對(duì)另一方產(chǎn)生影響以及會(huì)產(chǎn)生怎樣的影響呢？本研究擬建立C57BL／6小鼠胚胎干細(xì)胞系，著眼于將小鼠胚胎干細(xì)胞與小鼠惡性黑色素瘤B16細(xì)胞進(jìn)行共培養(yǎng)，探索共培養(yǎng)中兩種細(xì)胞相互作用情況，以及胚胎干細(xì)胞與腫瘤細(xì)胞共培養(yǎng)對(duì)腫瘤細(xì)胞生物學(xué)行為的影響。 目的 1.優(yōu)化小鼠胚胎成纖維細(xì)胞飼養(yǎng)層制備條件，以建立穩(wěn)定高效的C57BL／6小鼠胚胎成纖維細(xì)胞飼養(yǎng)層培養(yǎng)體系，用于小鼠胚胎干細(xì)胞的純化和建系培養(yǎng)。 2.建立穩(wěn)定的C57BL／6小鼠胚胎干細(xì)胞系。 3.通過(guò)小鼠胚胎干細(xì)胞與B16細(xì)胞體外共培養(yǎng)，及檢測(cè)共培養(yǎng)后B16細(xì)胞部分生物學(xué)行為變化，初步探討小鼠胚胎干細(xì)胞與B16細(xì)胞體外相互作用及其機(jī)制。 方法 1.取妊娠12.5-14.5d的胎鼠，組織消化法分離培養(yǎng)MEF；MTT法檢測(cè)MEF細(xì)胞增殖活性，篩選出制備飼養(yǎng)層中合適的絲裂霉素C作用MEF細(xì)胞的濃度時(shí)間、細(xì)胞接種密度及細(xì)胞代數(shù)。 2.妊娠3.5d的小鼠囊胚置于不同接種密度飼養(yǎng)層細(xì)胞培養(yǎng)體系上，觀察囊胚、內(nèi)細(xì)胞團(tuán)及ES細(xì)胞的生長(zhǎng)狀況，，進(jìn)一步驗(yàn)證優(yōu)化后MEF飼養(yǎng)層分離培養(yǎng)ES細(xì)胞的效果。 3.C57BL／6小鼠3.5d的囊胚置于小鼠胚胎成纖維細(xì)胞制作的飼養(yǎng)層上孵育，3-5d后分離內(nèi)細(xì)胞團(tuán)，擴(kuò)增傳至35代以上，觀察集落生長(zhǎng)情況，通過(guò)形態(tài)學(xué)、堿性磷酸酶染色、早期胚胎特異性抗原（SSEA-1）和OCT-4染色，及體內(nèi)分化實(shí)驗(yàn)對(duì)ES細(xì)胞集落進(jìn)行鑒定。 4.腫瘤細(xì)胞先接種后加入胚胎干細(xì)胞，胚胎干細(xì)胞先接種后加入腫瘤細(xì)胞，以及兩種細(xì)胞同時(shí)接種，采用ES條件培養(yǎng)基進(jìn)行共培養(yǎng)；及腫瘤細(xì)胞先接種后加入胚胎干細(xì)胞，采用DMEM／F12+20%FBS培養(yǎng)基進(jìn)行共培養(yǎng)，篩選合適的小鼠胚胎干細(xì)胞與B16細(xì)胞共培養(yǎng)體系。 5.通過(guò)小鼠胚胎干細(xì)胞與B16細(xì)胞體外共培養(yǎng)模型觀察小鼠胚胎干細(xì)胞與B16細(xì)胞共培養(yǎng)中相互作用，MTT法檢測(cè)共培養(yǎng)后B16細(xì)胞增殖能力及粘附能力的變化，transwell小室法檢測(cè)共培養(yǎng)后B16細(xì)胞侵襲及遷移能力的變化。 結(jié)果 1.絲裂霉素C10ug／ml作用3-3.5h、20ug／ml作用2-2.5h抑制MEF細(xì)胞效果較好；MEF以（1-2）×104／孔接種于96孔板，比以（3-6）×104／孔接種的細(xì)胞活力穩(wěn)定；絲裂酶素C處理MEF后1-4代比5、6代活力穩(wěn)定，活力維持時(shí)間較長(zhǎng)。 2.飼養(yǎng)層細(xì)胞不同接種密度培養(yǎng)體系下分析囊胚貼壁率、內(nèi)細(xì)胞團(tuán)孵出率及ES細(xì)胞克隆形成率，進(jìn)一步驗(yàn)證結(jié)果1中篩選出來(lái)的條件下制備的飼養(yǎng)層細(xì)胞囊胚貼壁率、內(nèi)細(xì)胞團(tuán)孵出率及ES細(xì)胞集落克隆形成率均較其他條件下高，最終可以分離培養(yǎng)出可穩(wěn)定傳代的ES細(xì)胞集落。 3.分離培養(yǎng)后得到的ES細(xì)胞可穩(wěn)定傳代至35代以上，且均呈集落樣生長(zhǎng)，AKP染色、免疫組化SSEA-1表面抗原和OCT-4抗原均為陽(yáng)性，接種于裸鼠及C57BL／6小鼠均可形成畸胎瘤，HE染色后均有三個(gè)胚層組織成分，符合小鼠ES細(xì)胞的特性。 4.四種不同條件下小鼠胚胎干細(xì)胞與B16細(xì)胞共培養(yǎng)，建立了合適的小鼠胚胎干細(xì)胞與B16細(xì)胞共培養(yǎng)體系。 5.小鼠胚胎干細(xì)胞與B16細(xì)胞共培養(yǎng)中小鼠胚胎干細(xì)胞能夠侵入并推開小鼠黑色素瘤B16細(xì)胞形成自己的生長(zhǎng)空間；與對(duì)照組比較共培養(yǎng)后腫瘤細(xì)胞的增殖能力、粘附能力、侵襲及遷移能力均顯著降低（P＜0.05，P＜0.01)。 結(jié)論 優(yōu)化條件后制備的小鼠胚胎成纖維細(xì)胞飼養(yǎng)層可以較好地支持囊胚貼壁、ICM孵出及ES細(xì)胞集落形成與生長(zhǎng)，獲得的ES細(xì)胞經(jīng)鑒定符合小鼠ES細(xì)胞的一般特性，穩(wěn)定傳至35代以上仍保持良好的胚胎干細(xì)胞干性特征，建立了穩(wěn)定的C57BL／6小鼠胚胎干細(xì)胞系。利用共培養(yǎng)實(shí)驗(yàn)?zāi)Ｐ�，進(jìn)行小鼠胚胎干細(xì)胞與B16細(xì)胞體外共培養(yǎng)，體外共培養(yǎng)中小鼠胚胎干細(xì)胞能夠侵襲腫瘤細(xì)胞并形成自己的生長(zhǎng)空間，并使B16細(xì)胞體外增殖能力、粘附能力、侵襲及遷移能力降低。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 尚克剛，胡新立，李子玉，王學(xué)慶，劉愛民，孟國(guó)良，童英;飼養(yǎng)層對(duì)維持新建ES細(xì)胞系的影響[J];北京大學(xué)學(xué)報(bào)(自然科學(xué)版);1994年04期

2 后曉南,譚毅,趙R

本文編號(hào)：1751976