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[1]全仁夫,鄭宣,許世超,等.大鼠毛囊干細胞體外培養(yǎng)及基因轉(zhuǎn)染方法[J].中醫(yī)正骨,2014,26(06):19-23.
　Zheng Xuan,Xu Shichao,Ni Yueming,et al.A method of hair follicle stem cells culture in vitro and gene transfection in rats Quan Renfu*[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2014,26(06):19-23.
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大鼠毛囊干細胞體外培養(yǎng)及基因轉(zhuǎn)染方法()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第26卷
期數(shù):
2014年06期
頁碼:
19-23
欄目:
基礎(chǔ)研究
出版日期:
2014-06-30

文章信息/Info

Title:
A method of hair follicle stem cells culture in vitro and gene transfection in rats Quan Renfu*
作者:
全仁夫1; 鄭宣2; 許世超1; 倪月明1; 謝尚舉2; 李長明2
1.浙江省杭州市蕭山區(qū)中醫(yī)院,浙江 杭州 311201; 2.浙江中醫(yī)藥大學,浙江 杭州 310012
Author(s):
Zheng Xuan; Xu Shichao; Ni Yueming; Xie Shangju; Li Changming.*
Xiaoshan Hospital of Traditional Chinese Medicine,Hangzhou 311201,Zhejiang,China
關(guān)鍵詞:
干細胞 毛囊 細胞培養(yǎng)技術(shù) 轉(zhuǎn)染 大鼠
Keywords:
Stem cells;  Hair follicle;  Cell culture techniques;  Transfection;  Rats
摘要:
目的:探討大鼠毛囊干細胞體外培養(yǎng)和基因轉(zhuǎn)染的方法。方法:采用組織塊法分離、培養(yǎng)大鼠觸須隆突部毛囊干細胞,經(jīng)Ⅳ型膠原差速貼壁法純化后,采用堿性磷酸酶染色和細胞免疫熒光染色進行細胞鑒定,測定細胞生長曲線,并分別采用脂質(zhì)體和慢病毒質(zhì)粒進行毛囊干細胞基因轉(zhuǎn)染。結(jié)果:經(jīng)兩次Ⅳ型膠原貼壁篩選后,毛囊干細胞呈克隆性生長,具有典型的干細胞生物學特征,堿性磷酸酶染色陽性,角蛋白-15、整合素-α6和整合素-β1表達均為陽性。細胞生長曲線顯示毛囊干細胞在接種后第3天時出現(xiàn)干細胞克隆; 第5天和第6天時細胞處于對數(shù)生長期。脂質(zhì)體介導的毛囊干細胞基因轉(zhuǎn)染率為1.55%～17.21%,中位數(shù)9.63%; 慢病毒介導的毛囊干細胞基因轉(zhuǎn)染率為62.14%～79.42%,中位數(shù)71.52%。結(jié)論:采用組織塊法分離、培養(yǎng)大鼠觸須隆突部毛囊干細胞,經(jīng)Ⅳ型膠原差速貼壁法純化后,毛囊干細胞可保持較好的生長狀態(tài)和較強的克隆形成能力,且慢病毒介導毛囊干細胞基因轉(zhuǎn)染比脂質(zhì)體轉(zhuǎn)染更高效、穩(wěn)定。
Abstract:
Objective:To explore the method of hair follicle stem cells(HFSCs)culture in vitro and gene transfection in rats.Methods:The HFSCs in palpus eminence of rats were isolated and cultured by using tissue explant method,and were purified by using type-Ⅳ collagen differential adherence method.Then,the cells were identified through alkaline phosphatase staining and cell immunofluorescence staining,and the cells growth curve were also determined,meanwhile,the gene transfection of HFSCs were conducted by using liposome and lentivirus plasmid respectively.Results:The HFSCs grew in clone after screening 2 times by type-IV collagen differential adherence,and they showed typical biological characteristics of stem cells.The alkaline phosphatase staining and the expression of keratin-15,integrin-α6 and integrin-β1 were all positive.The cells growth curve showed that HFSCs presented in clone at the 3rd day after inoculation,and the cells entered on the logarithmic growth phase at the 5th and 6th day after inoculation.The rate of HFSCs gene transfection mediated by liposome were 1.55% to 17.21% with a median of 9.63%,while the rate of HFSCs gene transfection mediated by lentivirus were 62.14% to 79.42% with a median of 71.52%.Conclusion:After isolated and cultured through tissue explant and purified through type-Ⅳ collagen differential adherence,the HFSCs in palpus eminence of rats can keep good growth state and strong ability clonality.Moreover,the HFSCs gene transfection conducted by lentivirus plasmid was more efficient and stable than that conducted by liposome.

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更新日期/Last Update: 1900-01-01