人结直肠活组织HIV-1黏膜感染模型的建立

目的 建立与HIV-1黏膜感染相关的结直肠活组织体外培养模型,利用假病毒模拟HIV-1进入黏膜建立感染的生物学过程. 方法 在知情同意下,取肿瘤手术患者癌旁正常结直肠组织,保留黏膜层与黏膜下层,钻取成统一大小(直径6 mm)的组织块置于12孔板中培养.HE染色观察培养0 d至13 d时组织形态结构的变化,噻唑蓝法(MTT法)同步检测组织活性.采用单轮复制周期假病毒感染黏膜并持续培养6～7 d,通过检测上清中p24含量验证该模型再现HIV-1黏膜感染的适用性;以进入临床实验的预防HIV-1黏膜感染的第1代微生物杀菌剂壬苯醇醚-9(N-9)为例,验证该模型对黏膜外用药物的反应性. 结果 HE染色观察至5 d时结直肠组织结构良好,13 d时黏膜组织结构依然可见.经MTT法检测黏膜培养4 d时活性高于80%,7 d的活性可保持在50%左右.假病毒感染该活组织模型后半量换液1、4、8 d的培养上清中,检测到p24的含量升高;同时,该组织模型活性可灵敏地反映出N-9浓度的变化. 结论 成功建立一种人体结直肠活组织HIV-1体外感染模型,可模拟人体HIV-1黏膜感染的生物学过程.

Abstract：

Objective To establish human colorectal tissue model in HIV-1 mucosal infection and by using pseudotyped virus to simulate the biological process of HIV-1 mucosal infection from HIV-1 entrying into mucosa to local infection establishment. Methods Tumor adjacent normal colorectal tissues were obtained with informed consent.After excised the muscularis externa,the mucosa and submucosa were dissected into the same blocks and cultured in 12-well cell culture plates.The cultured tissue structure and morphology were observed from day 0 to day 13 by staining with the hematoxylin eosin (HE),and the tissue activity was detected by 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The established tissues explants were infected by a single cycle replicated pseudotyped virus and propagated for 6-7 days,then subjected to the detection of p24 production within supernatant to verify the applicability of the model for the studying of HIV-1 mucosal infection.The applicability of the established explants for safety and reactivity evaluation of mucosa topical drugs was conducted by the using of first generation antiseptic Nonoxynol-9(N-9) as an example. Results HE staining showed the structure of colorectal tissue was remained well until 5th day and still evident until 13th day.The tissue activity of cultured mucosa was above 80% at day 4,and still remained over 50% at day 7 as detected by MTT assay.After infected by pseudo virus,the increased level of p24 was detected from supernatant collected on 1st,4th,8th day,which indicated a local infection was created.In addition,the dose changing of N-9 was reflected sensitively by the activity of this model. Conclusion Ex vivo human colorectal tissue model mimic HIV-1 mucosal infection was established that can be used to replicate the bioprocess of human HIV-1 mucosal infection.

Establishment of human colorectal tissue model in HIV-1 mucosal infection

Objective To establish human colorectal tissue model in HIV-1 mucosal infection and by using pseudotyped virus to simulate the biological process of HIV-1 mucosal infection from HIV-1 entrying into mucosa to local infection establishment. Methods Tumor adjacent normal colorectal tissues were obtained with informed consent.After excised the muscularis externa,the mucosa and submucosa were dissected into the same blocks and cultured in 12-well cell culture plates.The cultured tissue structure and morphology were observed from day 0 to day 13 by staining with the hematoxylin eosin (HE),and the tissue activity was detected by 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The established tissues explants were infected by a single cycle replicated pseudotyped virus and propagated for 6-7 days,then subjected to the detection of p24 production within supernatant to verify the applicability of the model for the studying of HIV-1 mucosal infection.The applicability of the established explants for safety and reactivity evaluation of mucosa topical drugs was conducted by the using of first generation antiseptic Nonoxynol-9(N-9) as an example. Results HE staining showed the structure of colorectal tissue was remained well until 5th day and still evident until 13th day.The tissue activity of cultured mucosa was above 80% at day 4,and still remained over 50% at day 7 as detected by MTT assay.After infected by pseudo virus,the increased level of p24 was detected from supernatant collected on 1st,4th,8th day,which indicated a local infection was created.In addition,the dose changing of N-9 was reflected sensitively by the activity of this model. Conclusion Ex vivo human colorectal tissue model mimic HIV-1 mucosal infection was established that can be used to replicate the bioprocess of human HIV-1 mucosal infection.