本文選題：人羊膜間充質(zhì)干細(xì)胞　切入點(diǎn)：人臍帶間充質(zhì)干細(xì)胞　出處：《鄭州大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景與目的: 腦膠質(zhì)瘤是顱內(nèi)最常見(jiàn)的腫瘤,雖然在腦膠質(zhì)瘤的分子、基因方面的研究已經(jīng)取得了許多進(jìn)步,但患者的預(yù)后仍不滿(mǎn)意,目前手術(shù)切除仍是腦腫瘤治療的首選方法,但根治性切除后復(fù)發(fā)率高是影響其長(zhǎng)期生存率的主要因素。而國(guó)內(nèi)外均有文獻(xiàn)報(bào)道顯示MSCs具有抑瘤特性,本實(shí)驗(yàn)組在之前的研究中已經(jīng)觀察到hMSCs可以抑制腫瘤細(xì)胞的生長(zhǎng)。另隨著C6大鼠膠質(zhì)瘤細(xì)胞系的成熟發(fā)展,研究人員認(rèn)為C6膠質(zhì)瘤細(xì)胞的相關(guān)性研究對(duì)于人腦膠質(zhì)瘤的基礎(chǔ)研究具有參考和指導(dǎo)價(jià)值。 本實(shí)驗(yàn)在建立人羊膜間充質(zhì)干細(xì)胞(human mesenchymal stem cells hMSCs)及人臍帶間充質(zhì)細(xì)胞(human umbilicalcord stem cells hUSCs)的體外培養(yǎng)和鑒定方法的基礎(chǔ)上,探討hMSCs及hUSCs對(duì)于C6膠質(zhì)瘤細(xì)胞不同共培養(yǎng)方法培養(yǎng)后可能出現(xiàn)的結(jié)果。在本研究中,我們將通過(guò)在體外實(shí)驗(yàn)中分別進(jìn)行hMSCs與hUSCs和C6共培養(yǎng),觀察hMSCs與hUSCs和C6的相互作用,進(jìn)一步明確hMSCs與hUSCs對(duì)C6生長(zhǎng)的影響的不同,以期為進(jìn)一步的研究提供參考和方向的選擇。 材料與方法:無(wú)菌條件下取正常足月剖腹產(chǎn)胎兒的羊膜和臍帶采用組織塊培養(yǎng)法及胰酶消化法分離并于含10%胎牛血清(fetal bovine serum,FBS)的MEM/F12培養(yǎng)基進(jìn)行培養(yǎng)。取P3代hMSCs及hUSCs采用免疫組織化學(xué)與流式細(xì)胞儀對(duì)其間充質(zhì)干細(xì)胞表面標(biāo)志CD29、CD44、HLA-ABC及CD29、CD44進(jìn)行鑒定和其細(xì)胞周期判定。取P3代的hMSCs及hUSCs,配制成濃度為1.0×10~6個(gè)/ml的直接共培養(yǎng)工作液與1.0×10~6個(gè)穩(wěn)定傳代3代以上C6單細(xì)胞懸液分別采用直接共培養(yǎng)和間接共培養(yǎng)的方法進(jìn)行共培養(yǎng),共分5組,羊膜直接共培養(yǎng)組(A組),羊膜間接共培養(yǎng)組(B組),臍帶直接共培養(yǎng)組(C組),臍帶間接共培養(yǎng)組(D組),空白對(duì)照組(E組),后光鏡下觀察細(xì)胞生長(zhǎng)情況,收集C6進(jìn)行流式細(xì)胞測(cè)定和電鏡超微結(jié)構(gòu)觀察。 結(jié)果:用酶消化法分離羊膜中的MSCs,可在體外進(jìn)行培養(yǎng),進(jìn)而通過(guò)貼壁分離法不斷得到純化。用酶消化法及組織塊培養(yǎng)法分離臍帶中的MSCs,可在體外進(jìn)行培養(yǎng),進(jìn)而通過(guò)貼壁分離法不斷得到純化。hMSCs及hUSCs免疫細(xì)胞化學(xué)染色顯示CD44和CD29均為陽(yáng)性反應(yīng):hMSCs及hUSCs的細(xì)胞周期分析具有干細(xì)胞的典型周期特性,流式細(xì)胞儀檢測(cè)傳代hMSCs陰性對(duì)照9.39%,CD29陽(yáng)性細(xì)胞比率為82.53%,CD44陽(yáng)性細(xì)胞比率為90.86%,HLA-ABC陽(yáng)性細(xì)胞比率為89.55%。流式細(xì)胞儀檢測(cè)傳代hUSCs陰性對(duì)照7.61%,CD29陽(yáng)性細(xì)胞比率為70.44%,CD44陽(yáng)性細(xì)胞比率為75.50%. 透射電鏡觀察見(jiàn)C6細(xì)胞不同程度出現(xiàn)細(xì)胞間連接消失,觀察像核分裂相減少,細(xì)胞體積縮小,細(xì)胞器空泡樣變及髓樣變改變,甚者出現(xiàn)細(xì)胞核固縮,出現(xiàn)典型細(xì)胞凋亡形態(tài),對(duì)照組E組細(xì)胞仍呈旺盛生長(zhǎng)狀態(tài)。 C6細(xì)胞中bcl-2隨時(shí)間延長(zhǎng)陽(yáng)性表達(dá)率呈下降趨勢(shì),不同時(shí)間比較差異具有顯著性(P＜0.01);C、D兩組較A、B兩組陽(yáng)性表達(dá)率降低趨勢(shì)更為明顯,且兩兩比較差異具有顯著性(P＜0.01);B、D兩組較A、C兩組陽(yáng)性表達(dá)率降低趨勢(shì)更為明顯(P＜0.01)。 C6細(xì)胞隨時(shí)間延長(zhǎng)細(xì)胞平均凋亡率呈上升趨勢(shì),不同時(shí)間比較差異具有顯著性(P＜0.01);C、D兩組較A、B兩組陽(yáng)性表達(dá)率上升趨勢(shì)更為明顯,且兩兩比較差異具有顯著性(P＜0.01);B、D兩組較A、C兩組陽(yáng)性表達(dá)率上升趨勢(shì)更為明顯(P＜0.01)。 結(jié)論: 1.Bcl-2在C6中呈高表達(dá)。 2.hMSCs與hUSCs能夠降低C6克隆團(tuán)細(xì)胞之間的黏附作用。 3.hMSCs及hUSCs與C6共培養(yǎng)后C6增殖能力減弱。 4.hMSCs及hUSCs與C6共培養(yǎng)后C6中bcl-2的表達(dá)隨時(shí)間延長(zhǎng)呈降低趨勢(shì)。 5.hMSCs及hUSCs與C6共培養(yǎng)后C6細(xì)胞凋亡率隨著時(shí)間延長(zhǎng)呈上升趨勢(shì)。 6.采用間接共培養(yǎng)方法共培養(yǎng)后C6細(xì)胞bcl-2的表達(dá)降低趨勢(shì)更為明顯。 7.hUSCs與C6共培養(yǎng)后C6細(xì)胞凋亡率表達(dá)上升趨勢(shì)更為明顯。
[Abstract]:Background and purpose:
Glioma is the most common intracranial tumors, although the molecules in glioma, genetic research has made many progresses, but the prognosis is still not satisfied with the current preferred method for surgical resection is the treatment of brain tumors, but the high recurrence rate after radical resection is the main factors affecting the long-term survival rate. But the domestic and foreign literatures have showed that MSCs has anti-tumor properties, in a previous study of the experimental group has been observed in the hMSCs can inhibit the growth of tumor cells. The other with the mature development of glioma cell line C6 rats, the researchers think the correlation of C6 glioma cells has reference value for based on the human brain glioma.
In this experiment, the establishment of human amniotic mesenchymal stem cells (human mesenchymal stem cells hMSCs) and human umbilical cord mesenchymal cells (human umbilicalcord stem cells hUSCs) based method for in vitro culture and identification of hMSCs and hUSCs, different in C6 glioma cells were cultured after culture method may occur as a result of. In this study, we will through respectively hMSCs and hUSCs and C6 in vitro co culture, the interaction effect of hMSCs and hUSCs and C6, hMSCs and hUSCs to further clarify the different effects on the growth of C6, to provide the reference and direction for further research.
Materials and methods: under aseptic conditions of normal full-term fetus amniotic membrane and umbilical cord culture method and enzymatic digestion and with 10% fetal bovine serum by tissue (fetal bovine serum, FBS) of the MEM/F12 medium. P3 hMSCs and hUSCs by immunohistochemistry and flow cytometry in mesenchymal stem cell surface markers CD29, CD44, HLA-ABC and CD29, were identified and the cell cycle of CD44. HMSCs and hUSCs determine P3, prepared into a concentration of 1 * 10~6 /ml direct co culture liquid and 1 * 10~6 passage 3 generation to C6 single cell suspension respectively. The direct co culture method and indirect co culture were co cultured, were divided into 5 groups, amniotic membrane in direct co culture group (group A), amniotic membrane in indirect co culture group (group B), umbilical cord direct co culture group (group C), umbilical cord indirect co culture group (D group) and control group (E group), light microscope The growth of the cells was observed, and C6 was collected for flow cytometry and ultrastructural observation of electron microscope.
Results: the digestive enzymes separation of amniotic MSCs can be cultured in vitro, then purified by adherent separation method has been used. Enzyme digestion and tissue culture method to separate the umbilical cord in MSCs can be cultured in vitro, and then through the wall of separation has been purified.HMSCs and hUSCs immunocytochemistry staining showed that CD44 and CD29 were positive reaction: the cell cycle of hMSCs and hUSCs analysis of the characteristics of a typical cycle with stem cell, serum hMSCs negative control 9.39% flow cytometry, CD29 positive cells ratio was 82.53%, CD44 positive cell rate was 90.86%, the rate of HLA-ABC positive cells of 89.55%. were detected by flow cytometry hUSCs negative control 7.61%, CD29 positive cells ratio was 70.44%, the rate of CD44 positive cells was 75.50%.
Transmission electron microscopy showed different degrees of C6 cell cell-cell junction disappeared, as observed mitoses are reduced, cell shrinkage, cell vacuolar degeneration and myeloid changed, what appeared karyopyknosis, typical apoptotic morphology, the control group E cells was still vigorous growth.
Bcl-2 C6 cells with time. The positive expression rate decreased, different time difference was statistically significant (P < 0.01); C, D two group compared with the A, two group of B positive expression rate decreased more obviously, and the 22 difference was statistically significant (P < 0.01); B, D two compared with group A, two group of C positive expression rate decreased more obviously (P < 0.01).
C6 cells with the prolonging of average cell apoptosis rate increased, the different time difference was statistically significant (P < 0.01); C, D two group compared with the A, B two group, the positive expression rate of upward trend is more obvious, and the 22 difference was statistically significant (P < 0.01); B, D two groups compared with A group, C two positive expression rate trend is more significant (P < 0.01).
Conclusion:
1.Bcl-2 is highly expressed in C6.
2.hMSCs and hUSCs can reduce the adhesion between C6 cloned cells.
The proliferation ability of C6 was weakened after co culture of 3.hMSCs and hUSCs with C6.
After co culture of 4.hMSCs and hUSCs and C6, the expression of Bcl-2 in C6 decreased with time.
After co culture of 5.hMSCs and hUSCs and C6, the apoptosis rate of C6 cells increased with time.
6. the decrease trend of bcl-2 expression in C6 cells was more obvious after co culture.
The increase of apoptosis rate in C6 cells was more obvious after co culture of 7.hUSCs and C6.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

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