人转化生长因子β3基因转染KLD-12自组装纳米肽纤维支架三维培养前软骨干细胞

Transfecting human transformation growth factor beta 3 gene into precartilaginous stem cells cultured in three-dimensional self-assembled peptide nanofiber scaffold of KLD-12

背景:作者前期试验证实了外源性细胞因子人转化生长因子β3具有很强的诱导前软骨干细胞向成熟软骨分化并促进其增殖的能力。国外的研究均证实了在自组装肽纳米纤维支架中三维培养有利于软骨源性干细胞的增殖与分化。目的:将人转化生长因子β3基因转染于三维培养于KLD-12肽纳米纤维支架内的前软骨干细胞中,并比较其与二维培养前软骨干细胞转染效率的差异。方法:以免疫磁珠分选法获取并纯化前软骨干细胞,将其种植于KLD-12肽纳米纤维支架内,使用xfectTM stem纳米干细胞转染试剂将人转化生长因子β3基因分别转染入KLD-12三维支架内培养的前软骨干细胞和普通二维培养基培养的前软骨干细胞,通过免疫荧光和反转录-聚合酶链反应法测定转染后48h不同组别转化生长因子β3基因的表达情况,并利用酶联免疫吸附法测定转染后不同时间细胞培养上清液中转化生长因子β3的质量浓度。结果与结论:免疫荧光结果显示,KLD-12三维支架内培养的前软骨干细胞转染后转化生长因子β3阳性细胞率明显高于普通二维培养基内前软骨干细胞(P<0.05),与反转录-聚合酶链反应法检测结果相同。酶联免疫吸附法测定结果显示,转染24h前软骨干细胞上清液中转化生长因子β3蛋白水平呈上升趋势,72h后达到峰值,随后稍下降进入平台期。提示人转化生长因子β3基因在三维培养于KLD-12肽纳米纤维支架内前软骨干细胞中的转染效率高于普通二维培养基培养的前软骨干细胞。

BACKGROUND：Previous tests of our research group have proved that,exogenous cytokines human transforming growth factor-β3（hTGF-β3） has a strong capacity of inducing precartilaginous stem cells（PSCs） differentiate to mature cartilage and of promoting the proliferation.Abroad studies also confirmed a beneficial effect on the proliferation and differentiation of cartilage-derived stem cells in three-dimensional culture of the self-assembled peptide nanofiber scaffold.OBJECTIVE：To transfect the hTGF-β3 gene in the PSCs cultured in three-dimensional self-assembled peptide nanofiber scaffold of KLD-12,and to compare the transfection efficiency with PSCs cultured in two-dimensional medium.METHODS：The PSCs were obtained and purified using magnetic-activated cell sorting method,then transplanted into the self-assembled peptide nanofiber scaffold of KLD-12.The gene of hTGF-β3 was respectively transfected into the PSCs cultured in self-assembled scaffold and common two-dimension medium by means of xfectTM stem nano-transfection reagent.After 48 hours,the gene expression levels of hTGF-β3 were detected by immunofluorescence and RT-PCR,and the concentration of TGF-β3 in the supernatant of medium was determined in different groups and different time points after transfection by the method of ELISA.RESULTS AND CONCLUSION：Immunofluorescence results showed that the rate of TGF-β3-positive cells in the group of PSCs cultured in three-dimensional self-assembled scaffold of KLD-12 was higher than those cultured in common two-dimensional medium（P〈0.05）,which was consistent with the test results of RT-PCR.By the method of ELISA,the concentration of TGF-β3 in the supernatant of medium increased at 24 hours after transected,and reached peak after 72 hours,then decreased a little and entered the plateau stage.It＇s indicated that the transfection efficiency of hTGF-β3 gene in PSCs cultured in self-assembled peptide nanofiber scaffold is higher than the group in common two-dimensional medium.