Construction and identification of lentiviral vector for RNA interference of human Versican gene and role of Versican in aggregative growth of dermal papilla cells

封明霞; 伍津津; 杨桂红

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Construction and identification of lentiviral vector for RNA interference of human Versican gene and role of Versican in aggregative growth of dermal papilla cells

更新时间：2026-01-09

- Construction and identification of lentiviral vector for RNA interference of human Versican gene and role of Versican in aggregative growth of dermal papilla cells
- Journal of Army Medical University Vol. 37, Issue 14, Pages: 1460-1465(2015)
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  CLC： R322.995;R394-33;R394.2
- Published：2015
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封明霞,伍津津,杨桂红.Versican基因RNAi慢病毒载体的构建及其对毛乳头细胞聚集性生长的干预作用[J].陆军军医大学学报(原第三军医大学学报),2015,37(14):1460-1465.

　Feng Mingxia,Wu Jinjin,Yang Guihong.Construction and identification of lentiviral vector for RNA interference of human Versican gene and role of Versican in aggregative growth of dermal papilla cells[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(14):1460-1465.
封明霞,伍津津,杨桂红.Versican基因RNAi慢病毒载体的构建及其对毛乳头细胞聚集性生长的干预作用[J].陆军军医大学学报(原第三军医大学学报),2015,37(14):1460-1465. DOI：

　Feng Mingxia,Wu Jinjin,Yang Guihong.Construction and identification of lentiviral vector for RNA interference of human Versican gene and role of Versican in aggregative growth of dermal papilla cells[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(14):1460-1465. DOI：

摘要

目的构建Versican基因RNAi慢病毒载体并研究Versican对毛乳头细胞聚集性生长的干预作用。方法根据人Versican基因序列，构建4个携带RNAi序列的慢病毒载体，利用293T细胞进行慢病毒包装。实验分为Control组（不做处理）、NC组（用空慢病毒载体包装的病毒转染细胞）和KD1、KD2、KD3、KD4 Versican RNAi慢病毒感染组。应用KD1、KD2、KD3、KD4 Versican RNAi慢病毒及NC慢病毒感染H1299细胞，荧光显微镜下观察感染效率，应用Real-time PCR检测Versican的表达，筛选出感染效率和特异性最高的慢病毒载体进行下一步研究。用慢病毒感染毛乳头细胞后，用Real-time PCR检测Versican基因的表达，并用Western blot检测Versican、ADAMTS4、p-c-JUN蛋白的表达，应用光镜观察毛乳头的生长情况。结果成功构建4种慢病毒载体，其中KD2慢病毒感染H1299细胞较其他慢病毒、Control和NC组显著下调Versican mRNA的表达(P0.01)。应用KD2慢病毒感染毛乳头细胞后，较Control和NC组明显下调毛乳头细胞Versican mRNA和蛋白的表达(P0.01)，ADAMTS4、p-c-JUN蛋白的表达较Control组明显下调(P0.01)，毛乳头细胞聚集性生长的特性消失。结论成功构建了Versican基因RNAi慢病毒载体。Versican基因通过c-JUN-ADAMTS-4通路调控毛乳头细胞的聚集性生长。

Abstract

Objective To construct lentiviral vector for RNA interference (RNAi) of Versican gene and study the role of Versican in aggregative growth of dermal papilla cells (DPCs). Methods According to human Versican mRNA sequence

4 specific RNAi sequences were designed for construction of 4 kinds of lentivirus vectors

and then the vectors were transferred into 293T cells for packaging lentiviruses. H1299 cells were divided into 6 groups

that is

a control group

a negative control group

and KD1

KD2

KD3 and KD4 groups transfected with KD1

KD2

KD3 and KD4 lentiviruses carrying different shRNAs

respectively. Transfection efficiency of lentiviruses was evaluated with a fluorescence microscope. The expression of Versican mRNA was detected by real-time PCR. Based on the detection data

the lentiviruses with high transfection efficiency and high specificity were chosen for further study. DPCs were transfected with the chosen lentiviruses

and the protein expression of Versican

ADAMTS4 and p-c-JUN was detected by Western blotting while the versican mRNA expression was assayed by real-time PCR. The characteristics of aggregative growth of DPCs was observed with a phase contrast microscope. Results Four kinds of Versican RNAi lentiviruses were constructed successfully

and KD2 lentiviruses showed the best transfection effect in the H1299 cells. The expression of Versican mRNA in the KD2 group was significantly decreased as compared with the other groups (P0.01). After the DPCs were transfected with KD2 lentiviruses

the expression of Versican at mRNA and protein levels was significantly lower than those of the control group and the negative control group (P0.01). In addition

the protein expression of ADAMTS4 and p-c-JUN was significantly decreased as compared with the control group (P0.01)

and the characteristic aggregative growth of DPCs disappeared. Conclusion The lentiviral vector for RNAi of Versican gene is constructed successfully. Versican plays a role in regulating the aggregative growth of DPCs via the c-JUN-ADAMTS4 pathway.

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