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[1]黃小龍,全仁夫,胡華輝,等.HLA-B2704基因型強(qiáng)直性脊柱炎患者來源的人誘導(dǎo)多能干細(xì)胞體系的建立[J].中醫(yī)正骨,2017,29(12):11-18.
　HUANG Xiaolong,QUAN Renfu,HU Huahui,et al.Establishment of human-induced pluripotent stem cells system originated from patients with HLA-B2704 genotypic ankylosing spondylitis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(12):11-18.
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HLA-B2704基因型強(qiáng)直性脊柱炎患者來源的人誘導(dǎo)多能干細(xì)胞體系的建立()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期數(shù):
2017年12期
頁碼:
11-18
欄目:
基礎(chǔ)研究
出版日期:
2017-12-20

文章信息/Info

Title:
Establishment of human-induced pluripotent stem cells system originated from patients with HLA-B2704 genotypic ankylosing spondylitis
作者:
黃小龍1; 全仁夫1; 胡華輝1; 李偉1; 李強(qiáng)1; 邱銳2; 孫亞萍3; 楊迪生4
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(xué),浙江 杭州 310053; 3.浙江大學(xué),浙江 杭州 310058; 4.浙江大學(xué)附屬第二醫(yī)院,浙江 杭州 310009
Author(s):
HUANG Xiaolong1; QUAN Renfu1; HU Huahui1; LI Wei1; LI Qiang1; QIU Rui2; SUN Yaping3; YANG Disheng4
1.Xiaoshan Hospital of Chinese Medicine,Hangzhou 311201,Zhejiang,China 2.Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 3.Zhejiang University,Hangzhou 310058,Zhejiang,China 4.The Second Affiliated Hospital of Zhejiang University,Hangzhou 310009,Zhejiang,China
關(guān)鍵詞:
脊柱炎; 強(qiáng)直性;  HLA抗原;  尿;  誘導(dǎo)多能干細(xì)胞
Keywords:
Key words spondylitis; ankylosing;  HLA antigens;  urine;  induced pluripotent stem cells
摘要:
目的:建立人類白細(xì)胞抗原(human leukocyte antigen,HLA)-B2704基因型強(qiáng)直性脊柱炎(ankylosing spondylitis,AS)患者來源的人誘導(dǎo)多能干細(xì)胞(human-induced pluripotent stem cells,hiPSCs)體系。方法:采用逆轉(zhuǎn)錄病毒介導(dǎo)的感染系統(tǒng)把OCT4、SOX2、c-MYC、KLF4這4個(gè)外源轉(zhuǎn)錄因子導(dǎo)入HLA-B2704基因型AS患者的尿液細(xì)胞中,重編程獲得hiPSCs。通過形態(tài)觀察、堿性磷酸酶染色、免疫熒光染色、內(nèi)源多能性相關(guān)基因檢測、體外擬胚體(embryoid bodies,EBs)分化檢測、EBs三胚層基因檢測以及畸胎瘤形成實(shí)驗(yàn),鑒定獲得的hiPSCs。結(jié)果:病毒感染尿液細(xì)胞后第4天至第5天細(xì)胞核質(zhì)比增大,第14天左右出現(xiàn)克隆,第24天后形成與人胚胎干細(xì)胞(human embryonic stem cells,hESCs)相似的克隆。堿性磷酸酶染色顯示,hiPSCs和hESCs-H1一樣,均被染成紫紅色,呈陽性。免疫熒光染色顯示,hESCs階段特異性胚胎抗原4及ESCs轉(zhuǎn)錄因子OCT4、SOX2均呈陽性。hiPSCs內(nèi)源多能性基因OCT4、SOX2、NANOG和REX1的表達(dá)與H1內(nèi)源多能性相關(guān)基因的表達(dá)比較,差異均無統(tǒng)計(jì)學(xué)意義(1.006±0.208,1.000±0.340,t=0.038,P=0.972; 1.061±0.140,1.000±0.387,t=0.431,P=0.689; 1.386±0.354,1.000±0.032,t=1.467,P=0.303; 1.280±0.283,1.000±0.013,t=1.398,P=0.235)。EBs高表達(dá)內(nèi)胚層基因AFP、GATA4,中胚層基因MEX1、TBX1,外胚層基因PAX6、SOX1; EBs三胚層基因AFP、GATA4、TBX1、MSX1、PAX6、SOX1的相對表達(dá)量均高于hiPSCs(60.695±8.746,1.000±0.245,t=9.647,P=0.011; 3.724±0.144,1.000±0.417,t=12.136,P=0.007; 4.224±0.869,1.000±0.130,t=4.988,P=0.038; 684.800±63.326,1.000±0.211,t=15.270,P=0.004; 131.561±15.785,1.000±0.232,t=11.700,P=0.007; 98.507±40.443,1.000±0.215,t=16.000,P=0.004)。hiPSC體內(nèi)形成畸胎瘤,HE染色可見內(nèi)、中、外3個(gè)胚層的組織細(xì)胞; 其中內(nèi)胚層為小腸上皮組織細(xì)胞,中胚層為肌肉組織細(xì)胞,外胚層為神經(jīng)上皮組織細(xì)胞。結(jié)論:采用逆轉(zhuǎn)錄病毒介導(dǎo)的感染系統(tǒng)把OCT4、SOX2、c-MYC、KLF4這4個(gè)外源轉(zhuǎn)錄因子導(dǎo)入HLA-B2704基因型AS患者的尿液細(xì)胞中,經(jīng)過重編程,可成功建立HLA-B2704基因型AS患者來源hiPSCs體系。
Abstract:
ABSTRACT Objective:To establish the human-induced pluripotent stem cells(hiPSCs)system originated from patients with human leukocyte antigen(HLA)-B2704 genotypic ankylosing spondylitis(AS).Methods:Four exogenous transcription factors including OCT4,SOX2,c-MYC and KLF4 were introduced into the urinary cells(UCs)of patients with HLA-B2704 genotypic AS by using retrovirus-mediated infection system,and the infected UCs were reprogrammed for obtaining hiPSCs.The hiPSCs were identified through morphologic observation,alkaline phosphatase staining,immunofluorescence staining,endogenous multipotent related gene detection,embryoid bodies(EBs)differentiation detection,EBs triploblastic gene detection and teratoma formation experiment.Results:The nuclear-cytoplasmic ratioincreased at the 4th and 5th day after UCs were infected by virus.The clone appeared at the 14th day and it was similar to human embryonic stem cells(hESCs)at the 24th day.The result of alkaline phosphatase staining showed that both hiPSCs and hESCs-H1 were presented with prunosus positive staining.The result of immunofluorescent staining showed that the stage specific embryonic antigen 4(SSEA4)of hESCs and the transcription factor of ESCs including OCT4 and SOX2 presented with positive staining.There was no statistical difference in the expression of endogenous multipotent related gene including OCT4,SOX2,NANOG and REX1 between hiPSCs and hiPSCs-H1(1.006+/-0.208 vs 1.000+/-0.340,t=0.038,P=0.972; 1.061+/-0.140 vs 1.000+/-0.387,t=0.431,P=0.689; 1.386+/-0.354,1.000+/-0.032,t=1.467,P=0.303; 1.280+/-0.283 vs 1.000+/-0.013,t=1.398,P=0.235).High expressions of endodermal genes including AFP and GATA4,mesodermal genes including MEX1 and TBX1 and ectodermal genes including PAX6 and SOX1 were found in EBs.The relative expression levels of triploblastic genes including AFP,GATA4,TBX1,MSX1,PAX6 and SOX1 were higher in EBs compared to hiPSCs(60.695+/-8.746 vs 1.000+/-0.245,t=9.647,P=0.011; 3.724+/-0.144 vs 1.000+/-0.417,t=12.136,P=0.007; 4.224+/-0.869 vs 1.000+/-0.130,t=4.988,P=0.038; 684.800+/-63.326 vs 1.000+/-0.211,t=15.270,P=0.004; 131.561+/-15.785 vs 1.000+/-0.232,t=11.700,P=0.007; 98.507+/-40.443 vs 1.000+/-0.215,t=16.000,P=0.004).Teratoma appeared in hiPSC.Small intestine epithelial histocytes,muscular histocytes and neuroepithelial histocytes were found respectively in entoderm,mesoderm and ectoderm of teratoma through HE staining.Conclusion:The hiPSCs system originated from patients with HLA-B2704 genotypic AS can be successfully established through introducing four exogenous transcription factors including OCT4,SOX2,c-MYC and KLF4 into UCs of patients with HLA-B2704 genotypic AS by using retrovirus-mediated infection system and reprogramming the UCs.

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備注/Memo

備注/Memo:
基金項(xiàng)目:浙江省科技計(jì)劃項(xiàng)目(2014C03031) 通訊作者:全仁夫 E-mail:[email protected]
更新日期/Last Update: 2018-05-02