發(fā)布時(shí)間：2018-01-07 13:05

  本文關(guān)鍵詞：人臍帶血間充質(zhì)干細(xì)胞移植治療大鼠急性心肌梗死的實(shí)驗(yàn)研究　出處：《廣州醫(yī)學(xué)院》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 急性心肌梗死 臍帶血 間充質(zhì)干細(xì)胞 移植 心功能

【摘要】：目的: 1.研究臍帶血來源的間充質(zhì)干細(xì)胞體外分離、純化及培養(yǎng)的條件,以建立穩(wěn)定的體外培養(yǎng)體系,滿足實(shí)驗(yàn)和臨床的需要。 2.探討臍血間充質(zhì)干細(xì)胞移植到心肌梗死大鼠體內(nèi),觀察其是否可以存活及對大鼠心功能的影響。 方法: 1.無菌條件下采取健康育齡產(chǎn)婦正常分娩胎兒的臍帶血42份,所有樣本均于采集8h內(nèi)分離。 2.臍帶血42份隨機(jī)分成3組: FBS(胎牛血清)未包被組,FBS包被組,Mesencult培養(yǎng)基組,每組12份。FBS未包被組和FBS包被組均使用含10%FBS的LG-DMEM培養(yǎng)基,Mesencult培養(yǎng)基組使用培養(yǎng)干細(xì)胞專用的Mesencult培養(yǎng)基,與其添加物配合使用。3組均用Ficoll淋巴細(xì)胞分離液,密度梯度法分離臍血單個(gè)核細(xì)胞(MNC),將單個(gè)核細(xì)胞按密度1 X 108/ml接種于T25培養(yǎng)瓶中 3.培養(yǎng)瓶置37℃飽和濕度含5%CO2孵育箱培養(yǎng),72h后全量換液,去除未貼壁細(xì)胞,以后均7天換液一次。待細(xì)胞長到80%鋪滿瓶底時(shí)結(jié)束原代培養(yǎng),按1:1進(jìn)行消化傳代。觀察不同培養(yǎng)條件對臍血MSCs生長的影響。 4.流式細(xì)胞術(shù)分析P2代細(xì)胞表面CD29、CD34、CD45、CD105標(biāo)志。 5.將36只SD大鼠隨機(jī)分成MSCs移植組、假手術(shù)組和心肌梗死組各12只。結(jié)扎左冠狀動(dòng)脈前降支制備大鼠心肌梗死模型, 1周后,經(jīng)尾靜脈注射帶DAPI標(biāo)記的臍血MSCs。 6.4周后測定大鼠血流動(dòng)力學(xué),并行免疫組織化學(xué)檢測移植細(xì)胞存活與分化情況及檢測梗死組織中FactorⅧ表達(dá)來比較三組微血管密度。 結(jié)果: 1.實(shí)驗(yàn)觀察顯示:經(jīng)典的MSCs培養(yǎng)方法,用未經(jīng)胎牛血清包被培養(yǎng)瓶培養(yǎng)的MNC很少出現(xiàn)貼壁,且大多為形態(tài)多樣的混雜細(xì)胞,無明顯的細(xì)胞集落形成。在少數(shù)標(biāo)本中約2周后細(xì)胞可達(dá)到融合,主要表現(xiàn)為大的扁圓形細(xì)胞、破骨細(xì)胞和梭形成纖維樣的MSCs,盡管這些混雜細(xì)胞原代培養(yǎng)也可以達(dá)到融合,但其擴(kuò)增能力明顯受到限制,且傳代未達(dá)2代。而臍血單個(gè)核細(xì)胞在合適的培養(yǎng)基中,以較高的密度種植于經(jīng)胎牛血清包被的培養(yǎng)瓶內(nèi),原代培養(yǎng)產(chǎn)生的貼壁細(xì)胞以間充質(zhì)干細(xì)胞為優(yōu)勢細(xì)胞,混雜有少量破骨樣細(xì)胞。而在Mesencult培養(yǎng)基下培養(yǎng),經(jīng)傳代后這些貼壁細(xì)胞可以被純化為生長良好的間充質(zhì)干細(xì)胞。其能保持良好的擴(kuò)增能力和均質(zhì)性。42份臍血中單個(gè)核細(xì)胞原代培養(yǎng),其中僅8份傳代培養(yǎng)成功,而其中Mesencult條件培養(yǎng)基有5份培養(yǎng)成功,明顯高于經(jīng)典培養(yǎng)方法。原代和P2代臍血MSCs的生長特性進(jìn)行觀察比較,原代MSCs在接種后的2-8d為生長的潛伏期,10-14d以后細(xì)胞進(jìn)入對數(shù)生長期,3-4周左右逐漸進(jìn)入平臺(tái)期,P2代臍血MSCs擴(kuò)增速度明顯快于原代培養(yǎng),2-4d倍增1次,5-10d后細(xì)胞進(jìn)入到對數(shù)生長期,10-14d進(jìn)入平臺(tái)期。 2.通過流式細(xì)胞儀檢測P2代的臍血MSCs結(jié)果顯示,P2代MSCs極弱表達(dá)CD34、CD45造血細(xì)胞標(biāo)志,穩(wěn)定地高表達(dá)CD29、CD105間充質(zhì)細(xì)胞相關(guān)的表面抗原。這與骨髓MSCs的表面抗原標(biāo)志相一致。表明臍血MSCs是存在于臍血中區(qū)別于造血細(xì)胞的一群處于未分化狀態(tài)的非定向干/祖細(xì)胞。 3.移植后4周,經(jīng)血流動(dòng)力學(xué)檢測,與心梗組比較,MSCs移植組左室心功能明顯改善(P0.05),移植組心肌組織中可以觀察到DAPI標(biāo)記細(xì)胞存在,但標(biāo)記細(xì)胞并未表達(dá)Troponin-T及connexin43,免疫組化染色檢測示MSCs移植組心肌微血管密度(MVD)明顯高于心梗組和假手術(shù)組。 結(jié)論 1.臍血單個(gè)核細(xì)胞以高密度(1X108cells/ml )接種在Mesencult培養(yǎng)基中、FBS包被培養(yǎng)瓶等條件下,可以在體外成功的培養(yǎng)出較純化的臍血MSCs,其培養(yǎng)成功率較高。臍血MSCs的免疫表型符合間充質(zhì)干細(xì)胞特征。 2.經(jīng)尾靜脈移植的臍血MSCs能存活在心肌梗死大鼠心肌組織中。 3.臍血MSCs移植能促進(jìn)心梗大鼠心功能恢復(fù),并刺激梗死部位血管生成。
[Abstract]:Objective:
1., we studied the isolation, purification and culture conditions of umbilical cord blood derived mesenchymal stem cells in vitro, so as to establish a stable in vitro culture system to meet experimental and clinical needs.
2. the umbilical cord blood mesenchymal stem cells were transplanted into the rats with myocardial infarction to observe whether it could survive and influence the cardiac function of rats.
Method:
1. under aseptic conditions, 42 umbilical cord blood samples from healthy childbearing age parturients were taken from normal childbirth, and all the samples were isolated in 8h.
2. 42 copies of umbilical cord blood were randomly divided into 3 groups: FBS (fetal bovine serum) non coated group, FBS group, Mesencult medium group, each group of 12.FBS coated group and FBS group were coated with 10%FBS LG-DMEM medium, Mesencult medium was used for stem cell culture special Mesencult medium and additives are used Ficoll with lymphocyte separation liquid using.3 group, mononuclear cells separated from umbilical cord blood by density gradient method (MNC), the mononuclear cells by density of 1 X 108/ml were seeded in T25 culture flask
3. of the culture flask containing 5%CO2 37 DEG C saturated humidity incubator culture, after 72h was all changed, the nonadherent cells were removed after 7 days, all was changed once. When the cells grow to 80% covered the bottom of the bottle at the end of primary culture, digestion and passage by 1:1. To observe the effects of different culture conditions on the growth of umbilical cord blood MSCs.
4. flow cytometry was used to analyze the CD29, CD34, CD45, and CD105 markers on the surface of P2 cells.
5. 36 SD rats were randomly divided into MSCs transplantation group, sham operation group and myocardial infarction group, 12 rats. The left anterior descending branch of left coronary artery was ligated to prepare rat myocardial infarction model. After 1 weeks, umbilical cord blood with DAPI labeled MSCs. was injected through tail vein.
Determination of hemodynamics in rats after 6.4 weeks, compared with three groups of parallel to microvessel density Factor VIII expression survival chemical detection and differentiation of transplanted cells and immunohistochemical detection in myocardial tissue.
Result:
1.: experimental observation shows that the culture method of the classic MSCs, without fetal bovine serum coated culture flask MNC rarely adherent, and mostly mixed cell morphological diversity, no obvious cell colony formation. In a few specimens in about 2 weeks after cell fusion can be achieved, the main performance for large the flat and round cells, osteoclasts and spindleformation fiber like MSCs, although these hybrid cells in primary culture can also achieve the fusion, but its ability was significantly restricted, and the passage is not up to the 2 generation. The umbilical cord blood mononuclear cells in a suitable medium to high density planting in the fetal bovine serum clear coated culture flask, primary culture of adherent cells to mesenchymal stem cells as the dominant cells, a small amount of osteoclast like cells mixed with. Cultured in the Mesencult culture medium, then the adherent cells can be purified for good growth of mesenchymal The stem cells can maintain good amplification ability and heterogeneity of.42 in umbilical cord blood mononuclear cells were cultured, which only 8 copies were cultured successfully, and the Mesencult conditioned medium 5 successfully cultured, was significantly higher than that of classical methods. The growth characteristics of cultured primary and P2 cells of umbilical cord blood MSCs were compared between the original. Generation of MSCs in 2-8d after inoculation for growth after incubation, 10-14d cells in logarithmic growth phase, 3-4 weeks gradually into the platform, the P2 generation of umbilical cord blood MSCs amplification significantly faster than the primary culture, 2-4d doubled 1 times, after 5-10d cells into the number of students for a long period of time, 10-14d into the platform.
2. by flow cytometry P2 generation of umbilical cord blood MSCs results showed that P2 MSCs very weak expression of CD34, CD45 hematopoietic cell markers, stable and high expression of CD29, CD105 mesenchymal cell associated surface antigens. This is consistent with the surface antigen of bone marrow MSCs sign. That is present in the umbilical cord blood MSCs iswho in a group of hematopoietic cells in non directional undifferentiated stem / progenitor cells.
4 3. weeks after transplantation, the hemodynamics, compared with myocardial infarction group, MSCs transplantation group significantly improved cardiac function of left ventricular (P0.05), transplantation group, myocardial tissue can be observed in DAPI labeled cells, but not the expression of Troponin-T and connexin43 labeled cells, immunohistochemical staining showed MSCs group myocardial microvascular density (MVD) was significantly higher than that in MI group and sham operation group.
conclusion
1. human umbilical cord blood mononuclear cells at high density (1X108cells/ml) were inoculated in Mesencult medium, FBS coated bottles and other conditions, can be successfully cultured with purified MSCs in umbilical cord blood in vitro, the success rate is high. The culture of umbilical cord blood MSCs immune phenotype of mesenchymal stem cells.
2. the umbilical cord blood MSCs transplanted through the tail vein can survive in the myocardium of rats with myocardial infarction.
3. MSCs transplantation in umbilical cord blood can promote the recovery of cardiac function in myocardial infarction rats and stimulate angiogenesis in the infarct site.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

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1 陸東風(fēng);吳昊;黃t，

本文編號(hào)：1392658