miR-26a对骨髓间充质干细胞增殖和分化的影响及对骨质疏松的作用机制

发布时间：2018-05-09 15:53

本文选题：microRNA-26a + 骨髓间充质干细胞　；参考：《辽宁医学院》2012年硕士论文

【摘要】：目的本实验根据实验室前期小鼠骨髓间充质干细胞芯片筛查结果拟利用小鼠骨髓间充质干细胞的成骨分化模型从基因、蛋白水平及体内实验,探讨miR-26a在成骨分化中的表达趋势及对骨髓间充质干细胞调控作用，并进一步探讨miR-26a对骨质疏松的作用机制。方法（1）采用卵巢切除法成功建OPM小鼠模型，使用全骨髓法分离培养骨质疏松小鼠的BMSCs(O-BMSCs)、假手术小鼠的BMSCs(S-BMSCs)和正常小鼠的BMSCs（N-BMSCs）。通过测定生长曲线、计算克隆形成率、流式细胞技术检测细胞表型分子对BMSCs进行初步干细胞鉴定；（2）利用成骨诱导液对骨髓间充质干细胞进行成骨诱导14d，实时定量聚合酶链反应（real time-PCR，RT-PCR）检测诱导3、7、10、14d后的miR-26a及成骨基因（Runx-2、OCN、Col-1）表达，同时利用Western blot技术进一步验证RT-PCR结果。（3）miR-26a在BMSCs中的功能实验,使用miR-26a的促进物miR-RiboTMmicroRNA-26a mimics及miR-26a抑制物miR-RiboTMmicroRNA-26a inhibitor通过siPORTTMNeoFXTMagent转染试剂瞬时转染BMSCs，3d后加入成骨诱导剂进行诱导培养,并同时设立转染microRNA-26a mimics NC的对照组。细胞培养7d、14d后,进行细胞形态、碱性磷酸酶染色、钙盐结节（茜素红染色）的检测；RT-PCR检测诱导10d后的miR-26a及成骨基因（Runx-2、OCN、Col-1）的表达变化。（4）提取OVX组及Sham组BMSCs进行培养，，RT-PCR、Western blot检测OVX组中miR-26a及成骨基因（Runx-2、OCN、Col-1）的表达。（5）miR-RiboTMmicroRNA-26a mimics复合HA/TACP材料植入裸鼠皮下及大鼠肾被膜下，两月后取材，HE切片检测成骨情况。结果（1）干细胞的分离与初步鉴定：成功培养小鼠N-BMSCs、O-BMSCs和S-BMSCs，生长曲线显示O-BMSCs的增殖能力低于S-BMSCs，克隆形成率显示O-BMSCs克隆形成率低于S-BMSCs的克隆形成率，成骨诱导后，ALP染色、茜素红染色和RT-PCR检测成骨分化能力显示O-BMSCs低于S-BMSCs；（2）miR-26a与BMSCs的成骨分化：RT-PCR检测在BMSCs向成骨分化过程中miR-26a的表达上升了约25倍，成骨基因表达也随着诱导天数的增加逐渐升高,差异有统计学意义（P0.05）。转染miR-26a mimics后的BMSCs在培养7d后细胞形态逐渐由梭形变为小短棒状，树枝状，无明显成纤维状，与阴性对照组类似；ALP染色显示试验组在培养7d就呈现强阳性，较对照组颜色深，茜素红染色显示试验组培养14d后即出现了数量较多的大小不等的深红色钙盐结节,而阴性对照组则出现较少、较零散的钙盐结节。RT-PCR检测上调miR-26a后成骨基因的表达均高于对照组,差异有统计学意义(P0.05)，Western Blot检测结果同RT-PCR。（3）OVX组别中miR-26a及成骨基因的表达：RT-PCR检测结果显示OVX组中miR-26a的表达低于Sham组4倍左右，成骨基因的表达也低于Sham组，差异有统计学意义(P0.05)，Western Blot结果同RT-PCR。（4）HE切片显示miR-26a mimics组成骨能力显著优于对照组。结论（1）OPM中的BMSCs表现为增殖活性降低、成骨分化能力降低的生物学特性。（2）本次实验首次验证了miR-26a与骨髓间充质干细胞成骨分化的关系，证实了miR-26a具有促进小鼠骨髓间充质干细胞成骨分化的作用。（3）miR-26a可能有恢复O-BMSCs的成骨能力，可能作为治疗OPM的新靶点，从而将骨代谢性疾病如骨质疏松症等发病机制进一步深化入干细胞及miRNAs水平。
[Abstract]:Purpose

Based on the results of bone marrow mesenchymal stem cell microarray screening , the expression of miR - 26a in osteogenic differentiation and the role of miR - 26a on bone marrow mesenchymal stem cells were investigated by using bone - forming differentiation model of mouse bone marrow mesenchymal stem cells . The mechanism of miR - 26a on osteoporosis was further discussed .

method

( 1 ) The mouse model was successfully constructed by the ovary resection method , and the bone marrow method was used to separate the bone marrow cells ( O - MSCs ) of the mouse with osteoporosis , the bone marrow cells of the sham - operated mice ( S - bone marrow cells ) and normal mice . The growth curve was measured , the clone formation rate was calculated , the flow cytometry was used to detect the cell phenotype molecules for the preliminary stem cell identification ;
( 2 ) The expression of miR - 26a and osteogenic genes ( Runx - 2 , OCN , Col - 1 ) after 3 , 7 , 10 and 14 days was detected by real time - polymerase chain reaction ( RT - PCR ) . The expression of miR - 26a and osteogenic genes ( Runx - 2 , OCN , Col - 1 ) was detected by Western blot .
The expression of miR - 26a and osteogenic genes ( Runx - 2 , OCN , Col - 1 ) after 10d was detected by RT - PCR . ( 4 ) The expression of miR - 26a and osteogenic genes ( Runx - 2 , OCN , Col - 1 ) was detected by RT - PCR and Western blot .

Results

( 1 ) Isolation and preliminary identification of stem cells : The growth curve showed that the proliferation ability of O - cells was lower than that of S - BMSC , and the growth curve showed that the growth rate of O - BMSC was lower than that of S - bone marrow , and the formation rate of the clones was lower than that of S - bone marrow . After osteogenic induction , ALP staining , alizarin red staining and RT - PCR were used to detect the bone differentiation ability .

( 2 ) The expression of miR - 26a was increased by about 25 times in the process of osteogenic differentiation by RT - PCR . The expression of miR - 26a increased gradually with the increase of induction days ( P0.05 ) .
Compared with the control group , the expression of the osteogenic gene was higher in the negative control group than in the control group ( P0.05 ) . The results of Western Blot were compared with RT - PCR .

( 3 ) The expression of miR - 26a and osteogenic genes in ovariectomized group : RT - PCR showed that the expression of miR - 26a was lower than that in Sham group , and the expression of osteogenic gene was lower than that in Sham group ( P0.05 ) . Western Blot analysis was similar to RT - PCR .

( 4 ) HE section showed that the ability of miR - 26a cells to make up bone was superior to that of the control group .

Conclusion

( 1 ) There was a decrease in proliferation activity and a decrease in osteogenic differentiation ability .

( 2 ) For the first time , the relationship between miR - 26a and bone marrow mesenchymal stem cells was verified , and it was confirmed that miR - 26a had the effect of promoting the differentiation of bone marrow mesenchymal stem cells into bone .

( 3 ) miR - 26a may have the ability to restore the osteogenic ability of O - cells , which may be used as a new target for the treatment , so that the pathogenesis of bone metabolic diseases such as osteoporosis can be further deepened into the stem cell and the miRNA level .

【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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