北京生物醫(yī)學(xué)工程

低氧微環(huán)境對(duì)P（3HB-co-4HB）與小鼠骨髓間充質(zhì)干細(xì)胞共培養(yǎng)形成心肌補(bǔ)片影響的實(shí)驗(yàn)研究

Experimental study on the effect of hypoxia microenvironment on the co-culture of P(3HB-co-4HB) and mouse bone marrow mesenchymal stem cells to form myocardial patch

作者：田琨穆軍升伯平周帆

單位：首都醫(yī)科大學(xué)附屬北京安貞醫(yī)院心臟外科，北京市心肺血管疾病研究所（北京 100029）解放軍總醫(yī)院第三醫(yī)學(xué)中心超聲科(北京 100039)

關(guān)鍵詞：低氧微環(huán)境； P（3HB-co-4HB）；心肌補(bǔ)片；心肌梗死

分類號(hào)：R318.04

出版年·卷·期（頁(yè)碼）：2019·38·3（227-234）

摘要：

目的探究低氧微環(huán)境對(duì)小鼠骨髓間充質(zhì)干細(xì)胞與P（3HB-co-4HB）材料共培養(yǎng)形成心肌補(bǔ)片的影響，為細(xì)胞移植術(shù)治療心肌梗死提供一種更有效的心肌補(bǔ)片。方法全骨髓培養(yǎng)法提取小鼠骨髓間充質(zhì)干細(xì)胞（mMSCs），取5代mMSCs流式細(xì)胞術(shù)鑒定表面抗原。將聚3羥基丁酸酯-co-4羥基丁酸酯[3-hydroxybutyrate-co-4-hydroxybutyrate，P(3HB-co-4HB)]與小鼠骨髓間充質(zhì)干細(xì)胞（mouse bone marrow mesenchymal stem cells，mMSCs）共培養(yǎng)制作成細(xì)胞補(bǔ)片，隨機(jī)分為常氧組和低氧組,每組各10個(gè)樣本，0、12、24 h分別用CCK-8法測(cè)定細(xì)胞增殖情況；掃描電子顯微鏡（scanning electron microscope，SEM）觀察補(bǔ)片存活、黏附、生長(zhǎng)情況。加入誘導(dǎo)劑5氮雜胞苷2周后，免疫熒光檢測(cè)兩組心肌肌鈣蛋白T(cTnT)的表達(dá)情況。結(jié)果在共培養(yǎng)24 h后，CCK-8法測(cè)定低氧組OD值(0.349±0.038)顯著大于常氧組（0.308±0.025）(n=10，P<0.05)，掃描電子顯微鏡觀察到低氧組P(3HB-co-4HB)材料上細(xì)胞數(shù)量更多，細(xì)胞與材料之間的黏附牢固，細(xì)胞形態(tài)正常。免疫熒光顯示低氧組cTnT表達(dá)比常氧組更加顯著。結(jié)論相對(duì)于常氧條件，低氧微環(huán)境可促進(jìn)骨髓間充質(zhì)干細(xì)胞在P（3HB-co-4HB）材料上的黏附、存活、增殖、分化，形成一種更有效的心肌補(bǔ)片。

Objective To investigate the effect of hypoxic microenvironment on the co-culture of mouse bone marrow mesenchymal stem cells with P(3HB-co-4HB) material to form a myocardial patch, and to provide a more effective myocardial for cell transplantation in the treatment of myocardial infarction. Methods Mouse bone marrow mesenchymal stem cells (mMSCs) were extracted by whole bone marrow culture. The surface antigens were identified by flow cytometry of 5 generations of mMSCs. 3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) and bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, mMSCs) co-cultured into cell supplements, randomly divided into normoxia group and hypoxia group, each group of 10 samples, 0, 12, 24 hours to determine cell proliferation by CCK-8 method; scanning electron microscope (scanning Electron microscope (SEM) was used to observe the survival, adhesion and growth of the patch. Two weeks after the addition of the inducer 5-azacytidine, the expression of cardiac troponin T (cTnT) was detected by immunofluorescence. Results After co-culture for 24 hours, the OD value of the hypoxic group measured by CCK-8 method (0.349±0.038) was significantly higher than that of the normoxic group (0.308±0.025) (n=10, P<0.05), which was observed by scanning electron microscopy. The number of cells in the oxygen group P(3HB-co-4HB) material is more, the adhesion between the cells and the material is firm, and the cell morphology is normal. Immunofluorescence showed that the expression of cTnT in the hypoxic group was more significant than in the normoxic group. Conclusions Compared with normoxic conditions, hypoxic microenvironment can promote the adhesion, survival, proliferation and differentiation of bone marrow mesenchymal stem cells on P(3HB-co-4HB) materials, and form a more effective myocardial patch.

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