目的 探讨脂肪间充质干细胞(ADMSCs)体外转染成为胰岛素分泌细胞的可能性及其在不同浓度葡萄糖环境下的胰岛素分泌情况.方法 以未转染的ADMSCs作为对照组,含有PcDNA3.1的ADMSCs作为空载体组,含有PcDNA3.1-hINS的ADMSCs作为重组载体组,转染后再将重组载体组根据培养时间不同分为1、6、12、18 d组,并将重组载体18 d组根据葡萄糖浓度的不同分为高糖组和低糖组.RT-PCR扩增人胰岛素基因,并构建含有人胰岛素基因的真核表达重组载体PcDNA3.1-hINS.经离心消化法获得ADMSCs,并通过流式细胞仪检测.瞬时转染,RT-PCR测各组胰岛素DNA的转录,ELISA法检测各组胰岛素分泌量,并对重组载体18 d组行葡萄糖刺激实验,计量资料以x±s表示,多组间的比较采用方差分析,两组比较采用t检验.结果 流式细胞仪检测ADMSCs表面抗原:CD44、CD90、CD106表达阳性,CD34、CD45、CD11b表达阴性.RT-PCR法检测重组载体组有胰岛素DNA转录,对照组和空载体组均无转录.ELISA法检测重组载体1、6、12、18 d组胰岛素分泌量分别为(4.7±0.8)mIU/L、(8.8±0.5)mIU/L、(8.9±0.8) mIU/L、(8.6±0.6) mIU/L,与对照组的(1.3±0.6) mIU/L和空载体组的(1.7±0.8)mIU/L分别比较,差异均有统计学意义(t=10.09,32.64,22.20,55.53；9.23,27.56,19.43,51.25,P＜0.05)；重组载体1d组与6、12、18 d组的胰岛素分泌量分别比较,差异有统计学意义(t=12.77,12.26,13.93,P＜0.05)；而6、12、18 d组间的胰岛素分泌量比较,差异无统计学意义(F=45.67,P＞0.05).高糖组和低糖组胰岛素分泌量比较,差异有统计学意义(t=2.03,P＜0.05).葡萄糖刺激实验阴性.结论 ADMSCs成功转染为胰岛素分泌细胞,并可以稳定分泌胰岛素,虽然胰岛素分泌量不能根据葡萄糖的浓度改变而改变,但仍为干细胞治疗糖尿病提供了一种新的种子细胞.
目的 探討脂肪間充質榦細胞(ADMSCs)體外轉染成為胰島素分泌細胞的可能性及其在不同濃度葡萄糖環境下的胰島素分泌情況.方法 以未轉染的ADMSCs作為對照組,含有PcDNA3.1的ADMSCs作為空載體組,含有PcDNA3.1-hINS的ADMSCs作為重組載體組,轉染後再將重組載體組根據培養時間不同分為1、6、12、18 d組,併將重組載體18 d組根據葡萄糖濃度的不同分為高糖組和低糖組.RT-PCR擴增人胰島素基因,併構建含有人胰島素基因的真覈錶達重組載體PcDNA3.1-hINS.經離心消化法穫得ADMSCs,併通過流式細胞儀檢測.瞬時轉染,RT-PCR測各組胰島素DNA的轉錄,ELISA法檢測各組胰島素分泌量,併對重組載體18 d組行葡萄糖刺激實驗,計量資料以x±s錶示,多組間的比較採用方差分析,兩組比較採用t檢驗.結果 流式細胞儀檢測ADMSCs錶麵抗原:CD44、CD90、CD106錶達暘性,CD34、CD45、CD11b錶達陰性.RT-PCR法檢測重組載體組有胰島素DNA轉錄,對照組和空載體組均無轉錄.ELISA法檢測重組載體1、6、12、18 d組胰島素分泌量分彆為(4.7±0.8)mIU/L、(8.8±0.5)mIU/L、(8.9±0.8) mIU/L、(8.6±0.6) mIU/L,與對照組的(1.3±0.6) mIU/L和空載體組的(1.7±0.8)mIU/L分彆比較,差異均有統計學意義(t=10.09,32.64,22.20,55.53；9.23,27.56,19.43,51.25,P＜0.05)；重組載體1d組與6、12、18 d組的胰島素分泌量分彆比較,差異有統計學意義(t=12.77,12.26,13.93,P＜0.05)；而6、12、18 d組間的胰島素分泌量比較,差異無統計學意義(F=45.67,P＞0.05).高糖組和低糖組胰島素分泌量比較,差異有統計學意義(t=2.03,P＜0.05).葡萄糖刺激實驗陰性.結論 ADMSCs成功轉染為胰島素分泌細胞,併可以穩定分泌胰島素,雖然胰島素分泌量不能根據葡萄糖的濃度改變而改變,但仍為榦細胞治療糖尿病提供瞭一種新的種子細胞.
목적 탐토지방간충질간세포(ADMSCs)체외전염성위이도소분비세포적가능성급기재불동농도포도당배경하적이도소분비정황.방법 이미전염적ADMSCs작위대조조,함유PcDNA3.1적ADMSCs작위공재체조,함유PcDNA3.1-hINS적ADMSCs작위중조재체조,전염후재장중조재체조근거배양시간불동분위1、6、12、18 d조,병장중조재체18 d조근거포도당농도적불동분위고당조화저당조.RT-PCR확증인이도소기인,병구건함유인이도소기인적진핵표체중조재체PcDNA3.1-hINS.경리심소화법획득ADMSCs,병통과류식세포의검측.순시전염,RT-PCR측각조이도소DNA적전록,ELISA법검측각조이도소분비량,병대중조재체18 d조행포도당자격실험,계량자료이x±s표시,다조간적비교채용방차분석,량조비교채용t검험.결과 류식세포의검측ADMSCs표면항원:CD44、CD90、CD106표체양성,CD34、CD45、CD11b표체음성.RT-PCR법검측중조재체조유이도소DNA전록,대조조화공재체조균무전록.ELISA법검측중조재체1、6、12、18 d조이도소분비량분별위(4.7±0.8)mIU/L、(8.8±0.5)mIU/L、(8.9±0.8) mIU/L、(8.6±0.6) mIU/L,여대조조적(1.3±0.6) mIU/L화공재체조적(1.7±0.8)mIU/L분별비교,차이균유통계학의의(t=10.09,32.64,22.20,55.53；9.23,27.56,19.43,51.25,P＜0.05)；중조재체1d조여6、12、18 d조적이도소분비량분별비교,차이유통계학의의(t=12.77,12.26,13.93,P＜0.05)；이6、12、18 d조간적이도소분비량비교,차이무통계학의의(F=45.67,P＞0.05).고당조화저당조이도소분비량비교,차이유통계학의의(t=2.03,P＜0.05).포도당자격실험음성.결론 ADMSCs성공전염위이도소분비세포,병가이은정분비이도소,수연이도소분비량불능근거포도당적농도개변이개변,단잉위간세포치료당뇨병제공료일충신적충자세포.
Objective To investigate the possibility of transfection of adipose-derived mesenchymal stem cells (ADMSCs) into the insulin-secreting cells in vitro,and assay the secretion of insulin of ADMSCs in high and low glucose environment.Methods The ADMSCs that untransfected were in the control group,the ADMSCs that contained PcDNA3.1 were in the vacant vector group,and the ADMSCs that contained PcDNA3.1-hINS were in the recombinant vector group.After transfection,the recombinant vector group were sub-divided into the 1,6,12,18 days groups.According to the concentrations of glucose,the recombinant vector 18 days group were divided into the high glucose group and low glucose group.Human insulin gene was amplified by RT-PCR,and the eukaryotic expression recombinant vector PcDNA3.1-hINS that contained the human insulin gene was constructed.The ADMSCs were obtained by digestion and centrifugation,and then underwent flow cytometry for identification.The transcription of insulin DNA was assayed by RT-PCR,and the levels of insulin were assayed by ELISA.Glucose test was done in the recombinant vector 18 days group.The measurement data was shown in the format of (x) ± s,the measurement data in multiple groups were compared by randomized analysis of variance,and the comparison among groups was performed by the t test.ResuIts The expressions of CD44,CD90,CD106 were positive,and the expressions of CD34,CD45 and CD11b were negative.No insulin DNA transcription was detected in the control group and vacant vector group.The levels of insulin secreted were (4.7 ± 0.8) mIU/L,(8.8 ± 0.5) mIU/L,(8.9 ± 0.8)mIU/L,(8.6 ± 0.6)mIU/L in the recombinant vector 1,6,12,18 days group,which were significantly higher than (1.3 ± 0.6) mIU/L in the control group and (1.7 ± 0.8) mIU/L in the vacant vector group (t =10.09,32.64,22.20,55.53 ; 9.23,27.56,19.43,51.25,P ＜ 0.05).There were significant differences in the levels of insulin secreted between the recombinant vector 1 day group and the recombinant vector 6,12,18 days groups (t =12.77,12.26,13.93,P ＜0.05).There were no significant difference in the levels of insulin secreted between the recombinant vector 6,12,18 days groups (F =45.67,P ＞ 0.05).There was a significant difference in the level of insulin secreted between the high glucose group and the low glucose group (t =2.03,P ＜ 0.05).The result of the glucose stimulation test was negative.Conclusion The ADMSCs are transfected into insulinsecreting cells in vitro successfully,and the secretion of insulin is stable.Although the secretion of insulin can't change in line with the concentration of glucose,it is a new seed cell for the treatment of diabetes with stem cells.
