目的 观察携带降钙素基因相关肽(CGRP)的慢病毒对细胞生存和分化的影响. 方法 培养大鼠骨髓间充质干细胞(MSCs),携带CGRP的慢病毒载体转染MSCs(MSCsCGRP++组),空病毒载体转染MSCs作为对照组;应用流式细胞仪检测病毒转染率,酶联免疫吸附(ELISA)法测定CGRP蛋白表达,应用流式细胞仪及细胞免疫组化检测细胞表面标记物,胎盘蓝染色观察细胞存活能力,噻唑蓝(MTT)法检测细胞活力,β-半乳糖苷酶染色检测细胞衰老情况. 结果 携带CGRP的慢病毒转染MSCs后1d,荧光显微镜未观察到细胞有荧光,但ELISA测定MSCsCGRP+ +组有少量CGRP表达,随着转染时间延长,细胞逐渐出现荧光,慢病毒转染MSCs后3d和4d,镜下可见强荧光表达,细胞转染率分别为(77.87％和79.58％),ELISA检测MSCsCGRP++组CGRP表达量较对照组[(19.53±0.50) pg/ml和(3.12±0.001) pg/ml]增加(t=48.964,P＜0.01).慢病毒转染MSCs后3d,流式细胞仪检测MSCs仍高表达CD29和CD90,CD31的表达为24.75％,细胞转染后7d,CD31的表达为20.72％,与对照组6.63％比较CD31表达增加,vWF表达也随着转染时间延长表达逐渐增加;细胞转染后3d,MSCsCGRP+/+组及对照组均未见α-SMA表达,细胞转染14 d,在MSCsCGRP++组α SMA染色仍阴性,对照组胞浆见α-SMA阳性表达;细胞转染后3d和7d,MTT及台盼蓝染色结果显示两组间细胞活力及存活情况均无差异性(3d细胞活力t=-0.253,3d细胞存活率t=-0.307,7d细胞活力t=0.290,7d细胞存活率t=-0.690,均P＞0.05),β半乳糖苷酶检测细胞衰老程度在两组间差异也无统计学意义(3 d t=0.167,7 dt=0.141,均P＞0.05),随着转染时间延长,细胞转染14d,MSCsCGRP++组衰老程度较对照组降低(t=2.446,P＜0.05). 结论 转染慢病毒后细胞生物活性无明显影响,细胞仍具有增殖分化能力,细胞转染分泌的CGRP能促进细胞向内皮分化,抑制细胞向平滑肌细胞分化,这为基因工程细胞疗法治疗血管增殖性疾病提供新的思路和理论及实验依据.
目的 觀察攜帶降鈣素基因相關肽(CGRP)的慢病毒對細胞生存和分化的影響. 方法 培養大鼠骨髓間充質榦細胞(MSCs),攜帶CGRP的慢病毒載體轉染MSCs(MSCsCGRP++組),空病毒載體轉染MSCs作為對照組;應用流式細胞儀檢測病毒轉染率,酶聯免疫吸附(ELISA)法測定CGRP蛋白錶達,應用流式細胞儀及細胞免疫組化檢測細胞錶麵標記物,胎盤藍染色觀察細胞存活能力,噻唑藍(MTT)法檢測細胞活力,β-半乳糖苷酶染色檢測細胞衰老情況. 結果 攜帶CGRP的慢病毒轉染MSCs後1d,熒光顯微鏡未觀察到細胞有熒光,但ELISA測定MSCsCGRP+ +組有少量CGRP錶達,隨著轉染時間延長,細胞逐漸齣現熒光,慢病毒轉染MSCs後3d和4d,鏡下可見彊熒光錶達,細胞轉染率分彆為(77.87％和79.58％),ELISA檢測MSCsCGRP++組CGRP錶達量較對照組[(19.53±0.50) pg/ml和(3.12±0.001) pg/ml]增加(t=48.964,P＜0.01).慢病毒轉染MSCs後3d,流式細胞儀檢測MSCs仍高錶達CD29和CD90,CD31的錶達為24.75％,細胞轉染後7d,CD31的錶達為20.72％,與對照組6.63％比較CD31錶達增加,vWF錶達也隨著轉染時間延長錶達逐漸增加;細胞轉染後3d,MSCsCGRP+/+組及對照組均未見α-SMA錶達,細胞轉染14 d,在MSCsCGRP++組α SMA染色仍陰性,對照組胞漿見α-SMA暘性錶達;細胞轉染後3d和7d,MTT及檯盼藍染色結果顯示兩組間細胞活力及存活情況均無差異性(3d細胞活力t=-0.253,3d細胞存活率t=-0.307,7d細胞活力t=0.290,7d細胞存活率t=-0.690,均P＞0.05),β半乳糖苷酶檢測細胞衰老程度在兩組間差異也無統計學意義(3 d t=0.167,7 dt=0.141,均P＞0.05),隨著轉染時間延長,細胞轉染14d,MSCsCGRP++組衰老程度較對照組降低(t=2.446,P＜0.05). 結論 轉染慢病毒後細胞生物活性無明顯影響,細胞仍具有增殖分化能力,細胞轉染分泌的CGRP能促進細胞嚮內皮分化,抑製細胞嚮平滑肌細胞分化,這為基因工程細胞療法治療血管增殖性疾病提供新的思路和理論及實驗依據.
목적 관찰휴대강개소기인상관태(CGRP)적만병독대세포생존화분화적영향. 방법 배양대서골수간충질간세포(MSCs),휴대CGRP적만병독재체전염MSCs(MSCsCGRP++조),공병독재체전염MSCs작위대조조;응용류식세포의검측병독전염솔,매련면역흡부(ELISA)법측정CGRP단백표체,응용류식세포의급세포면역조화검측세포표면표기물,태반람염색관찰세포존활능력,새서람(MTT)법검측세포활력,β-반유당감매염색검측세포쇠로정황. 결과 휴대CGRP적만병독전염MSCs후1d,형광현미경미관찰도세포유형광,단ELISA측정MSCsCGRP+ +조유소량CGRP표체,수착전염시간연장,세포축점출현형광,만병독전염MSCs후3d화4d,경하가견강형광표체,세포전염솔분별위(77.87％화79.58％),ELISA검측MSCsCGRP++조CGRP표체량교대조조[(19.53±0.50) pg/ml화(3.12±0.001) pg/ml]증가(t=48.964,P＜0.01).만병독전염MSCs후3d,류식세포의검측MSCs잉고표체CD29화CD90,CD31적표체위24.75％,세포전염후7d,CD31적표체위20.72％,여대조조6.63％비교CD31표체증가,vWF표체야수착전염시간연장표체축점증가;세포전염후3d,MSCsCGRP+/+조급대조조균미견α-SMA표체,세포전염14 d,재MSCsCGRP++조α SMA염색잉음성,대조조포장견α-SMA양성표체;세포전염후3d화7d,MTT급태반람염색결과현시량조간세포활력급존활정황균무차이성(3d세포활력t=-0.253,3d세포존활솔t=-0.307,7d세포활력t=0.290,7d세포존활솔t=-0.690,균P＞0.05),β반유당감매검측세포쇠로정도재량조간차이야무통계학의의(3 d t=0.167,7 dt=0.141,균P＞0.05),수착전염시간연장,세포전염14d,MSCsCGRP++조쇠로정도교대조조강저(t=2.446,P＜0.05). 결론 전염만병독후세포생물활성무명현영향,세포잉구유증식분화능력,세포전염분비적CGRP능촉진세포향내피분화,억제세포향평활기세포분화,저위기인공정세포요법치료혈관증식성질병제공신적사로화이론급실험의거.
Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide (CGRP) gene on cells biological activity and differentiation of rat bone marrow stem cells(MSCs).Methods Rat MSCs were isolated and cultured by granulocytes adherent.MSCs were transfected with Lenti-EGFP CGRP(MSCsCGRP+/+ group),While MSCs were transfected with Lenti-EGFP as control group.Cell transfection rate was detected by flow cytometry,protein secretion in the above-mentioned MSCsCGRP+ + supernatant was detected using ELISA method.Cells surface markers weare detected by flow cytometry and immunohistochemistry.Trypan blue was used to examin the survive rate,β galactosidase staining was used to examin aging of MSCs transfection,and MTT was used to examine cell vitality.Results At first day after transfecting with Lenti-EGFP-CGRP,fluorescence was not observed by fluorescence microscope,but a small amount of CGRP protein was detected by ELISA in MSCsCGRP+/+ group,at 3 days and 4 days after transfecting with MSCs,strong fluorescence was observed by fluorescence microscope (the cell transfection rates were 77.87％ and 79.58％).The CGRP expression was significantly higher in MSCsCGRP+ + group than in control group [(19.53±0.50) pg/ml vs.(3.12±0.00) pg/ml,t=48.964,P＜0.01].At three days after transfection with MSCs,CD29 and CD90 expression were significantly higher,as compared with control group,CD31 expression was increased in MSCsCGRP+ /+ group.Seven days after transfection with MSCs,CD31 expression was significantly increased in MSCsCGRP+ + group,vWF expression was significantly increased in MSCsCGRP+ + group after MSCs were transfected with LentiEGFP CGRP for 14 days,but a SMA expression was decreased in MSCsCGRP+ +group.At 3 days and 7 days after transfection with Lenti-EGFP-CGRP,the proliferation,survive and aging showed no difference in MSCsCGRP+/+group and in control group (the proliferation of cell:t=0.253,0.290the survive of cell t=-0.307,0.690,all P＞0.05).At 14 days after transfection with Lenti-EGFP-CGRP,aging of cell were decreased in MSCsCGRP+ + group as compared with control group (t=2.446,P＜ 0.05).Conclusions After MSCs are transfected with Lenti EGFP-CGRP,biological characteristics of MSCs has no significant effects,there is still proliferation and differentiation activity.Cell secretion of CGRP can promote the endothelial cell differentiation,and inhibit the differentiation to smooth muscle cells.The CGRP modification of MSCs may play a role in the regulation of angiogenesis.
