艾滋病猴模型的免疫活化及青蒿琥酯对其干预作用的研究

发布时间：2018-05-06 10:36

本文选题：SIV + 中国恒河猴　；参考：《广州中医药大学》2017年博士论文

【摘要】：目的:对比不同进展类型SIV感染中国恒河猴T淋巴细胞免疫活化水平,线粒体抗病毒蛋白水平,探索长期不进展型SIV感染猴的内在病理机制。通过对不同进展类型SIV感染中国恒河猴的SIVmac239反复攻毒,探索长期不进展型感染猴免疫活化水平的稳定性,以及反复感染对正常进展型和长期不进展型SIV感染猴的影响。通过观察青蒿琥酯对SIV感染中国恒河猴免疫活化水平的干预作用,从调节免疫异常激活的角度寻找中医药干预艾滋病的治疗策略。方法:1.不同进展类型SIV感染中国恒河猴模型免疫特征对比研究:将17只中国恒河猴按照是否感染,感染后血浆病毒载量,以及感染后存活时间,分为健康对照(healthy control,HC)组;典型进展(typical progressor,TP)组;长期不进展(viremiccontrollor,VC)组;精英控制(elite controllor,EC)组。检测免疫活化调节相关指标,两周后重复检测以验证相关指标的稳定性。2.中国恒河猴SIV重复攻毒研究:SIV感染中国恒河猴8只,其中正常进展型4只,精英控制型4只。4只正常进展型恒河猴随机分为攻毒组和对照组,每组2只;4只精英控制型恒河猴随机分为攻毒组和对对照组,每组2只。攻毒组在第0周、第4周和第8周以5MID100(5倍100%感染剂量)静脉注射SIVmac239病毒悬液1 mL。3.青蒿琥酯对SIV感染中国恒河猴模型干预研究:健康中国恒河猴8只,复制艾滋模型,待进展到平台期,分为2组,青蒿琥酯治疗组4只,生理盐水对照组4只。青蒿琥酯连续给药8周,分别于给药前,给药后2周、4周、6周、8周,停药后4周、8周检测免疫活化调节相关指标。结果:1.不同进展类型SIV感染中国恒河猴模型免疫特征对比研究结果TP组病毒载量为典型的潜伏期水平,平均值为lg 5.25 copies/ml。VC组较TP组低(P0.05),平均值为lg4.02 copies/ml。EC组病毒载量长期处于测不出的水平,即小于50copies/ul。HC组、TP组和EC组白细胞计数相近,VC组高于其他三组。HC组CD4细胞比例最高,三个感染组之间未见明显差异,都略低于HC组。CD4计数是TP组最低,略低于其他三组。CD8细胞比例四组恒河猴未见明显差异。HC组CD4/CD8比值高于其他三个感染组,TP、VC和EC组CD4/CD8比值无差异。四个组中,TP单核细胞比例平均数最高,其次是VC组和EC组,HC组最低。单核细胞计数是VC组最高,其次是TP组和EC组,HC组最低。HC组恒河猴CD4细胞表面CD69表达水平维持在较低水平,其他三组感染猴都有不同程度的升高。TP组CD69水平最高,明显高于其他三个组,与HC组之间差异最大(P0.01),而VC组和EC组低于TP组,但是高于HC组。四组恒河猴CD4细胞表面CD25表达水平的相对情况与CD69类似,TP组最高(与HC组比较P0.01),VC组次之(与HC组比较P0.05)。HC猴CD8细胞的CD69表达水平较低,而另外三组恒河猴则有所升高。CD8细胞CD25表达水平与CD69情况相似,HC组水平较低而其他三组都略有升高,TP组升高最明显。TP型恒河猴和VC型恒河猴CD4细胞的CD38表达水平高于HC猴。EC型恒河猴CD38表达水平低于TP组(P0.01)和VC组(P0.01)。EC组CD4细胞HLA-DR表达与TP组(P0.05)和VC组(P0.05)差异显著。EC组CD38表达比例低于HC猴,平均值在10%以下,但是两组差异无统计学显著性。EC组CD8细胞CD38表水平TP组(P0.01)和VC组(P0.05)差异显著。EC组CD8细胞表达HLA-DR表达与TP组(P0.01)和VC组(P0.01)比较有统计学差异。三个感染组恒河猴CD4细胞PD-1表达水平相近,HC组略高于三个感染组。CD8细胞的PD-1表达水平也是三个感染组水平相似,但是相较HC组有所下降,且下降幅度大于CD4细胞结果。组间比较在统计学上无显著性。三个感染组SIV特异性CD8细胞比值都有所下降,其中EC组下降最少,其次是VC组。TP猴SIV特异性CD8比值下降最严重,与HC猴差异显著(P0.01)。EC组明显高于TP组(P0.01)。SIV特异性CD8细胞绝对值,TP组低于HC组,而VC组和EC组则高于HC组,其中EC组最高,但统计学上无显著性。SIV特异性CD8细胞的PD-1表达水平,VC和EC两个长期不进展组与HC在同一水平,TP组均数高于其他三组。SIV感染中国恒河猴CD8细胞CD38表达水平和HLA-DR表达水平均与SIV特异性CD8细胞比值成反比。VC和EC两个长期不进展组Treg比值与HC组基本一致,而TP显著高于另外三个组,差异有统计学意义。Treg绝对数在四组恒河猴中未见明显差异,TP组均值略高于其他三组。在全部17只恒河猴中,Treg的Ki67表达率显著高于非Treg CD4细胞,前者中位数大约是后者的12倍。在全部4组恒河猴的CD4细胞中,CTLA-4在Treg上的表达率明显高于在非Treg细胞上的表达比例。VC和EC两个长期不进展组相比HC组和TP组,CTLA-4表达水平都有下调,TP和VC、EC组之间差异显著(P0.05)。CTLA-4在非Treg上的表达水平,TP高于其他三组。2.中国恒河猴SIV重复感染实验研究正常进展型恒河猴TP-A在第一次攻毒后出现乏力、畏寒、消瘦,在第22天死亡,尸体行系统解剖,未见明显异常。其余恒河猴未见明显症状。精英控制猴EC-A病毒载量无变化,EC-B在第三次攻毒后上升至可测水平,但水平保持在3以下,维持至少8周,在第三次攻毒后16周之前恢复至测不出水平。TP-A猴在第一次攻毒后白细胞明显下降,第2周达到最低点,第3周有所回升。TP-B猴在第二次攻毒后白细胞有所上升,持续3周后恢复到实验前水平。实验第16周后有所下降。EC-A和EC-B在重复攻毒后白细胞出现反复波动,最高水平达12×103/μl,最低低至12×103/μl,第三次攻毒4周后趋于稳定。TP-B猴在二次攻毒后淋巴细胞比值波动增大,第三次攻毒后缓慢上升。EC-A和EC-B在第一次和第三次攻毒后淋巴细胞比值有所波动,波动结束后数值高于实验前。TP-B猴纯真CD4细胞比值在第一次攻毒后波动较大,第二次和第三次攻毒后波动明显减小,但数值都略低于攻毒前。TP-A猴在第一次攻毒后CD4细胞增殖水平上升,2周后下降。TP-B猴纯真CD4细胞在第一次和第二次攻毒后低于反复攻毒前水平。TP-B纯真CD8细胞在第二次攻毒后明显低于实验前。TP-A猴在第一次攻毒后Treg比值大幅上升。TP-A猴Treg细胞CTLA-4表达水平在第一次攻毒后迅上升,2周后达到最高点后开始下降。TP-A猴在第一次攻毒后CD4细胞CD38表达水平迅速大幅上升,第3周达到实验前大约3倍水平,而TP-B猴在每次攻毒后出现下降。TP-A猴CD4细胞HLA-DR表达水平在第一次攻毒后持续上升,直至第3周。TP-B猴CD8细胞CD38表达水平在第二次攻毒1周后大幅上升,随后维持在较高水平维持相对波动。TP-A猴在第一次攻毒后CD8细胞HLA-DR表达水平迅速大幅下降直至第3周。3.青蒿琥酯治疗SIV感染中国恒河猴模型的实验研究给药8周内,白细胞计数从第4周开始下降,第6周降至最低点后开始回升。停药后白细胞继续上升,停药4周回升至下降前水平。给药第6周淋巴细胞比例上升至最高点,明显高于对照组(P0.05),随后下降。给药8周内,治疗组嗜中性粒细胞比例从第4周开始出现下降,至第6周明显低于对照组(P0.05),后缓慢回升。给药组CD8细胞比例和计数在停药后较对照组低,其中停药4周和8周CD8比例的差异有统计学意义(P0.05)。在给药4周,给药组CD8细胞的CD69表达水平明显低于对照组(P0.05)。给药后,治疗组CD8细胞HLA-DR和CD38表达水平开始下降,到第4周两者都明显低于对照组(P0.05)。该两项指标从第6周开始回升,两组差异消失。给药2周后HLA-DR+CD8细胞计数下降百分比明显大于对照组(P0.01),停药4周和8周下降百分比也大于对照组(P0.05)。给药第2周和停药第4周,CD38+CD8细胞计数下降百分比都大于对照组(P0.05)。给药后4周,CD8细胞CD107a表达下降。结论:结论一:长期不进展型较正常进展型有更完善的CD8细胞功能。CD4/CD8比值不能评价SIV感染中国恒河猴的病程进展类型。长期不进展型SIV感染中国恒河猴CD4 T淋巴细胞和调节性T细胞活化明显低于正常进展猴,其中精英控制型恒河猴CD8 T淋巴细胞活化明显低于正常进展猴。CD8细胞异常激活水平越高,对SIV具有杀伤性的细胞比例就越低。存在除了抗病毒免疫以外的其他抑制淋巴细胞异常激活的机制。结论二:精英型SIV感染中国恒河猴淋巴细胞免疫水平的稳定性较典型进展型恒河猴强。重复攻毒可抑制TP猴CD4细胞和CD8细胞更新,加速TP猴病情进展,可使EC型恒河猴病毒量一过性上升。。结论三:青蒿琥酯可改善SIV感染中国恒河猴细胞毒性T细胞免疫异常激活。青蒿琥酯治疗中出现的嗜中性粒细胞下降是可逆的。
[Abstract]:Objective: To compare the immune activation level of T lymphocyte in Chinese Ganges RIver monkeys with different progressive types of SIV, the level of mitochondrial antiviral protein and explore the internal pathological mechanism of long term non progressive SIV infected monkeys. Through repeated attack on the SIVmac239 of different progressive type SIV infected Ganges RIver monkeys in China, the immune activation of chronic infective monkeys was explored. Level stability, and the effect of repeated infection on normal progressing and long-term unprogressing SIV infected monkeys. Through the observation of the intervention of artesunate to the immune activation level of SIV infected Ganges RIver monkeys in China, from the angle of regulating the activation of immune abnormality, the treatment strategy of interfering with AIDS was found. Method: 1. different SIV sense of different progression types Comparative study on immune characteristics of Ganges RIver monkey model: 17 Chinese Ganges RIver monkeys were divided into healthy control (healthy control, HC) group according to whether infection, post infection plasma viral load, and the survival time after infection; typical progress (typical progressor, TP) group; long term non progression (viremiccontrollor, VC) group; elite control (Elite controllor,) EC group. Detection of immune activation regulation related indicators, two weeks after repeated tests to verify the stability of the related indicators.2. Chinese Ganges RIver monkey SIV repeat attack study: SIV infected 8 Chinese monkeys, of which 4 were normal progressing, 4 of elite control type.4 normal progressive Ganges RIver monkeys were divided into the attack group and the control group, 2 in each group; 4 Elite control The Ganges RIver monkey was randomly divided into the attack group and the control group, with 2 rats in each group. In the zeroth week, fourth and eighth weeks, the SIVmac239 virus suspension of 1 mL.3. artesunate was injected with 5MID100 (5 times the 100% infection dose) to the intervention study of the Chinese Ganges RIver monkey model infected by SIV: 8 healthy Chinese Ganges RIver monkeys, replicating the AIDS model, and waiting for the platform stage, 2 groups were divided into 2 groups: artesunate treatment group 4, saline control group 4. Artesunate was given for 8 weeks, respectively, before administration, 2 weeks, 4 weeks, 6 weeks, 8 weeks after administration, 4 weeks after stopping, 8 weeks to detect immune activation regulation related indexes. Results: the immune response of TP group of Ganges RIver monkey model in different types of advanced types of advanced type 1. infection For the typical latent period level, the average value of LG 5.25 copies/ml.VC group was lower than that of the TP group (P0.05), the average value of the lg4.02 copies/ml.EC group was not found for a long time, that is, less than the 50copies/ul.HC group, the white cell count of TP and EC groups was similar, and the VC group was higher than the other three groups of.HC group, and the three infected groups were not found. The significant difference was slightly lower than group HC.CD4 count is the lowest in group TP, slightly lower than the proportion of other three groups of.CD8 cells in four groups of Ganges RIver monkeys, no significant difference in.HC group CD4/CD8 ratio is higher than the other three infection groups, TP, VC and EC group CD4/CD8 ratio no difference. Four groups, TP mononuclear ratio is the highest, followed by VC group and group, the lowest. Mononuclear group. Cell count was the highest in group VC, followed by group TP and group EC. The CD69 expression level of CD4 cell surface in the lowest.HC group of group HC was maintained at a lower level. The other three groups of infected monkeys had the highest level of CD69 in the.TP group, obviously higher than the other three groups, and the difference between the HC group and the HC group (P0.01), while the VC group and the group were lower than those in the.TP group, but higher but higher than those in the group. In group HC, the relative level of CD25 expression on the surface of CD4 cells in four groups of Ganges RIver monkeys was similar to that of CD69. Group TP was the highest (compared with HC group, P0.01), VC group was lower (compared with HC group, P0.05).HC monkey CD8 cell expression level was lower, while the other three groups were higher than those in the other groups. The level of CD38 expression in the CD4 cells of the.TP Ganges RIver monkey and VC type Ganges RIver monkey was higher than that of the HC monkey.EC type Ganges RIver monkey. The expression level of the CD38 expression level was lower than that of the TP group (P0.01) and the VC group (P0.01). In the two groups, there was no significant difference in the CD8 cell CD38 surface level TP group (P0.01) and VC group (P0.05) in group.EC, and the expression of CD8 cell HLA-DR expression in.EC group was significantly different from that in TP group (P0.01) and that of the three group. The expression level of Ganges RIver monkey cells was similar, and the group was slightly higher than that of the three infected groups. The level of PD-1 expression was also similar in the three infection groups, but compared with the HC group, the decrease was greater than that of the CD4 cells. The comparison between the groups was not statistically significant. The ratio of SIV specific CD8 cells in the three infected groups decreased, and the EC group decreased the least, followed by the SIV specific CD8 ratio of the.TP monkey in the VC group was the most serious, and the most serious. The difference of HC monkey significantly (P0.01) was significantly higher than that of group TP (P0.01).SIV specific CD8 cell absolute value, TP group was lower than group HC, while VC and EC group was higher than HC group, which was the highest in group.EC, but there was no significant difference between two groups of long period non progression and other three. The CD38 expression level and HLA-DR expression level of group.SIV infected Ganges RIver monkey CD8 cells were inversely proportional to the ratio of SIV specific CD8 cells to.VC and EC, the Treg ratio was basically consistent with the HC group, while TP was significantly higher than the other three groups. There was no significant difference between the absolute number of the four groups of the four groups of Ganges RIver monkeys. The value of Treg was significantly higher than that of the other three groups. The Ki67 expression rate of Treg was significantly higher than that of non Treg CD4 cells, the median of the former was about 12 times that of the latter. In all 4 groups of Ganges RIver monkey CD4 cells, the expression rate of CTLA-4 in Treg was significantly higher than that in the non Treg cell expression.VC and EC two long-term non progression groups compared to the HC group and the HC group. TP group, the expression level of CTLA-4 was down, TP and VC, the difference between EC group was significant (P0.05) the expression level of.CTLA-4 in non Treg, TP higher than the other three groups of.2. Chinese monkey SIV repeated infection experimental study of normal progressive Ganges RIver monkey TP-A in the first attack after the first attack of fatigue, fear cold, emaciation, died in twenty-second days, the body was dissected, the body dissection, no systematic anatomy, not seen There was no obvious symptom in the other Ganges RIver monkeys. The elites control monkey EC-A virus load did not change, the EC-B increased to the measurable level after the third attack, but the level remained below 3, maintained at least 8 weeks, and recovered to the undetected.TP-A monkey before the first attack of poison after the third attack, and the leukocyte decreased significantly after the first attack of poison, and reached second weeks. The lowest point, third weeks after the recovery of.TP-B monkeys after second attacks of white blood cells increased, after 3 weeks after the recovery to the pre experiment level. After sixteenth weeks, the experiment decreased.EC-A and EC-B after repeated attacks of leukocytes repeated fluctuations, the highest level of 12 x 103/ Mu L, the lowest to 12 * 103/ Mu L, third times after 4 weeks of attack to stabilize.TP-B monkey The fluctuation of lymphocyte ratio increased after the two attack of poison. After the third attack, the ratio of.EC-A and EC-B fluctuated at the first and third times of attack. The value of the pure CD4 cell ratio of the.TP-B monkey was higher than that before the experiment. The fluctuation of the ratio of the pure CD4 cells after the first attack was larger, and the fluctuation of the second and third attack after the attack was obviously reduced. Small, but the value is slightly lower than before the first attack of.TP-A monkey CD4 cell proliferation level after the first attack, 2 weeks after the decrease of.TP-B monkey pure CD4 cells after the first and second attacks were lower than before repeated attack of.TP-B pure CD8 cells after second attacks were significantly lower than before the first attack of the Treg ratio of the.TP-A monkey after the first attack on the Treg ratio. The expression level of CTLA-4 in the Treg cell of.TP-A monkey increased rapidly after the first attack. After 2 weeks, the expression level of CD38 in the CD4 cell of the.TP-A monkey increased rapidly after the first attack, and the level of the CD38 was about 3 times before the experiment, while the TP-B monkey decreased the CD4 cell HLA-DR expression level in.TP-A monkey after each attack. After the first attack of poison, the CD38 expression level of CD8 cells in.TP-B monkeys increased significantly after the second attack for 1 weeks, and then maintained at a relatively high level of relative fluctuation in the HLA-DR expression level of CD8 cells in CD8 cells after the first attack of poison to the third weeks of.3. artesunate for SIV infection in China's Ganges RIver monkey model. In the 8 weeks, the white blood cell count began to decline from fourth weeks and to the lowest point after sixth weeks. After the drug stopped, the leukocyte continued to rise and the drug was stopped for 4 weeks to the pre descending level. The proportion of lymphocytes increased to the highest point for sixth weeks, obviously higher than the control group (P0.05), and then decreased. The treatment group was neutrophils within 8 weeks. The ratio of cells decreased from fourth weeks to sixth weeks, obviously lower than that of the control group (P0.05), and then slowly recovered. The proportion and count of CD8 cells in the administration group were lower than those of the control group, of which the difference in the proportion of CD8 in 4 weeks and 8 weeks was statistically significant (P0.05). The expression level of CD8 cells in the administration group was significantly lower than that of the control group for 4 weeks (P0 .05). After administration, the expression level of HLA-DR and CD38 in the CD8 cells in the treatment group began to decrease, and both were significantly lower than the control group at fourth weeks (P0.05). The two indexes began to rise from sixth weeks and the two groups disappeared. The percentage of HLA-DR+CD8 cell counts decreased significantly after 2 weeks of Administration (P0.01), and the percentage of the decrease in 4 and 8 weeks was greater than that of the control group. The percentage of CD38+CD8 cell counts decreased more than that of the control group (P0.05) for second weeks and fourth weeks. Conclusion: the CD107a expression of CD8 cells decreased at 4 weeks after administration. Conclusion: a better CD8 cell function.CD4/CD8 ratio of long-term non progression type than normal progression type can not evaluate the progression of SIV infection of Ganges RIver monkeys in China. The activation of CD4 T lymphocyte and regulatory T cell in Chinese Ganges RIver monkey was significantly lower than that of normal progressing monkey. The activation of CD8 T lymphocyte in the elite controlled Ganges RIver monkey was significantly lower than that of normal progressing monkey.CD8 cells, the higher the abnormal activation level of.CD8 cells, the lower the percentage of cytotoxic cells to SIV. Other mechanisms other than immunization to inhibit the abnormal activation of lymphocyte. Conclusion two: the stability of lymphocyte immunity in Ganges RIver monkey is stronger than that of typical progressive Ganges RIver monkey. Repeated attack can inhibit the renewal of CD4 and CD8 cells in TP monkey and accelerate the progression of TP monkey, which can increase the amount of EC type Ganges RIver monkey virus in one over sex. Three: artesunate can improve the activation of immune abnormalities in the cytotoxic T cells of SIV infected Ganges RIver monkeys in China. The neutrophils decline in artesunate treatment is reversible.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R259;R-332

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