中国汉族CHC患者病程及对规范化治疗反应性与KIR2DL2基因相关性的群体分析

发布时间：2018-05-21 20:32

本文选题：中国汉族人群 + CHC　；参考：《华中科技大学》2013年博士论文

【摘要】：目的 1、通过观察中国汉族人群中CHC患者和健康人之间KIR基因、HLA-C基因的频率变化,深入探讨中国汉族人群中KIR基因、HLA-C基因与CHC病程的相关性; 2、通过观察中国汉族人群中CHC患者经规范化治疗后不同反应者的KIR基因HLA-C基因的频率变化,寻找对CHC治疗反应有影响力的基因； 3、经荟萃分析(Meta-analysis)对pubmed上多个CHC治疗效果研究资料进行分析和概括,并结合本次研究结果对KIR2DL2对CHC治疗反应的影响； 4、通过调节KIR2DL2基因的表达,观察其对NK细胞功能影响的特点,分析KIR2DL2影响CHC治疗反应的可能机制,为CHC治疗以及预后判断提供可能途径和新的治疗靶点。方法 1.入选中国汉族人群中CHC病例通过生化指标检测ALT, ELISA检测HCV抗体,Realtime-PCR定量检测HCV RNA。ELISA检测排除入选病例HAV、HBV、HDV、 HEV、HIV、病毒感染。 2.通过饱和盐析的方法从研究对象的抗凝血中提取DNA,采用序列特异性引物扩增技术(PCR—Sequence Specific Primer, PCR-SSP)对样本进行KIR和HLA-C分型,读取结果后计算基因频率,并统计分析比较中国汉族人群CHC患者和健康对照者之间的差异。 3.中国汉族人群中CHC患者接受规范化治疗,即聚乙二醇干扰素-α(1.5微克/公斤体重/周)和利巴韦林(0.9g/天)。所有患者进行至少6个月治疗后按照与以前报道的病毒学标准分为两个治疗结果：持续病毒学应答(sustained virological responders,SVR)和无应答(nonresponders, NR),提取研究对象DNA,采用PCR-SSP对样本进行KIR和HLA-C分型,统计分析比较SVR和NR之间的差异。 4.引入配对分析方法,将KIR2DL1与HLA-C2、KIR2DL2与HLA-C1配对,统计配对频率,比较中国汉族人群CHC患者和健康对照者之间、SVR和NR之间的差异。 5.在pubmed数据库(1995.11-2013.4)中,以‘'KIR2DL2"和“HCV”为关键词搜索文献,并按照需要英文书写、与国际标准相同的CHC诊断标准和治疗方法及结果判断、PCR-SSP检测KIR基因并含有KIR2DL2频率数据,患者除HCV感染外无其他疾病等筛选条件,选出合适的文献用于Meta分析,确定文献异质性、发表偏倚,选择固定效应模型计算总体风险系数。 6.使用经5-Aza-dc处理后的NK92细胞,该细胞表面可高表达KIR2DL2分子,观察不同抗体浓度抗体封闭KIR2DL2受体后,NK细胞对K562细胞的杀伤活性。 7. SPSS17.0对数据进行统计分析。结果 1.基因分型结果表明：KIR2DL2、KIR2DS3计算的OR值均大于1,提示在CHC患者中这两种基因的频率高于健康对照组,这可能是CHC发生的危险因素；同时,HLA-Cw*06, HLA-Cw*15, KIR2DS4计算的OR值小于1,提示在CHC患者中这些基因的频率低于健康对照组,这些基因的存在可能可以抑制CHC的发生。 2.在SVR与NR患者的分型结果中,我们可以看到KIR2DL2和HLA-Cw*03计算的OR值仍然大于1,提示在治疗效果较差的CHC患者(NR)中KIR2DL2基因的频率高于SVR组,携带有该基因的CHC患者的治疗反应要弱。 3. KIR/HLA-C配对分析显示,KIR2DL2+HLA-C group1配对OR值大于1,同时该组合在NR患者中的频率要高于SVR,提示在CHC中这种组合的存在可能有利于CHC的发生以及不利于CHC的治疗；KIR2DL1+HLA-C group2配对OR值小于1,提示这种组合的存在可能对抑制CHC的发生有一定作用。 4. Meta分析结果同样提示KIR2DL2对CHC治疗的负面影响。 5.经5-Aza-dc处理后的NK92细胞,该细胞表面可高表达KIR2DL2分子,在使用抗体浓度20μg/ml封闭KIR2DL2受体后,NK细胞对K562细胞的杀伤活性明显增强。结论 1.KIR2DL2可以抑制NK细胞活性； 2. KIR2DL2和它的受体HLA-C groupl可能有利于中国汉族人群中CHC的发生以及对PeglFN-a与利巴韦林联合治疗的反应不佳； 3. KIR2DS3在中国汉族人群CHC患者中的频率高于健康对照组,这可能是中国汉族人群中CHC发生的危险因素； 4. HLA-Cw*06, HLA-Cw*15, KIR2DS4CHC患者中的频率低于健康对照组,这些基因的存在可能可以抑制CHC的发生。本研究的创新点 1.目前,在高加索人群中有关于KIR与CHC相关性的报道,但在中国汉族人群中未见相关报道,本研究主要通过PCR-SSP检测中国汉族人群CHC患者中KIR频率与疾病以及治疗的相关性。 2.通过研究结果及Meta分析,首次提出在中国汉族人群中KIR2DL2为CHC发生以及治疗的负面因子。本研究的意义 1.通过研究,发现在中国汉族人群中KIR2DL2的存在具有较强的NK细胞抑制能力,使得KIR2DL2的存在可能不利于HCV的清除,导致HCV感染规范化治疗效果差,进而有利于HCV持续性感染发生。 2. K1R2DL2可成为一种HCV治疗效果预判指标,也是中国汉族人群中CHC发生流行病学危险因素,在后期研究中,KIR2DL2可成为免疫治疗CHC的重要靶点。
[Abstract]:objective
1, by observing the frequency changes of the KIR gene and HLA-C gene between CHC patients and healthy people in Chinese Han population, the correlation between the KIR gene, HLA-C gene and the course of CHC in the Chinese Han population was investigated.
2, by observing the changes in the frequency of the KIR gene HLA-C gene of the CHC patients in the Chinese Han population after the standardized treatment of the KIR gene, the genes that have influence on the response to the treatment of CHC were found.
3, Meta-analysis was used to analyze and summarize the results of multiple CHC therapy on PubMed, and the effect of this study on the response of KIR2DL2 to CHC treatment.
4, through the regulation of the expression of KIR2DL2 gene, the characteristics of its effect on the function of NK cells were observed, and the possible mechanisms of the effect of KIR2DL2 on the response of CHC therapy were analyzed. The possible pathways and new therapeutic targets for the treatment of CHC and the prognosis of the treatment were provided.
Method
In 1. Chinese Han population, CHC cases were detected by biochemical indexes, ALT, ELISA and HCV were detected by ELISA. HCV RNA.ELISA detection was used to detect HAV, HBV, HDV, HEV, HIV, and virus infection by Realtime-PCR quantitative detection of HCV RNA.ELISA.
2. DNA was extracted from the anticoagulant of the subjects by the method of saturated salting out, and the sequence specific primer amplification (PCR Sequence Specific Primer, PCR-SSP) was used for the KIR and HLA-C typing of the samples. After reading the results, the gene frequency was calculated, and the differences between the Chinese Han population CHC patients and the healthy controls were compared and analyzed.
In 3. Chinese Han population, CHC patients received standardized treatment, namely peginterferon - alpha (1.5 microgram / kilogram weight / week) and Leigh Bhave Lin (0.9g/ days). All patients were divided into two treatments according to the previously reported virology criteria after at least 6 months of treatment: sustained virological responders, SVR And nonresponders (NR), DNA was extracted. PCR-SSP was used to classify KIR and HLA-C samples, and the differences between SVR and NR were analyzed statistically.
4. paired analysis was introduced to match the KIR2DL1 with HLA-C2, KIR2DL2 and HLA-C1, to compare the paired frequencies, and to compare the differences between the CHC patients and the healthy controls in the Han population of the Chinese Han population, and between the SVR and the NR.
5. in the PubMed database (1995.11-2013.4), "'KIR2DL2" and "HCV" for the search of the literature, and in English written in need, the same as the international standard CHC diagnostic criteria and treatment methods and results, PCR-SSP detection of the KIR gene and the KIR2DL2 frequency data, patients with the exception of HCV infection without other diseases and other screening conditions, selected. Appropriate literature is used for Meta analysis, identifying literature heterogeneity, publishing bias, and choosing fixed effect models to calculate the overall risk coefficient.
6. the NK92 cells treated by 5-Aza-dc were used to express the KIR2DL2 molecules on the surface of the cells, and the killing activity of NK cells to K562 cells was observed after the antibody concentration of the antibody was closed to the KIR2DL2 receptor.
7. SPSS17.0 statistical analysis of the data.
Result
The results of 1. genotyping showed that the OR values calculated by KIR2DL2 and KIR2DS3 were all greater than 1, suggesting that the frequency of these two genes in CHC patients was higher than that in the healthy control group. This may be a risk factor for the occurrence of CHC; at the same time, the OR values of HLA-Cw*06, HLA-Cw*15 and KIR2DS4 were less than 1, suggesting that the frequencies of these genes in CHC patients were lower than those in the healthy control group. The presence of these genes may inhibit the occurrence of CHC.
2. in the typing results of patients with SVR and NR, we can see that the OR value calculated by KIR2DL2 and HLA-Cw*03 is still greater than 1, suggesting that the frequency of KIR2DL2 gene in CHC patients with poor therapeutic effect (NR) is higher than that of the SVR group, and the therapeutic response of the CHC patients with this gene is weak.
3. KIR/HLA-C pairing analysis showed that the KIR2DL2+HLA-C group1 paired OR value was greater than 1, and the frequency of this combination in NR patients was higher than SVR, suggesting that the presence of this combination in CHC may be beneficial to the occurrence of CHC and not to the treatment of CHC; KIR2DL1+HLA-C group2 pairing OR value is less than 1, suggesting that the presence of this combination may inhibit the existence of this combination. There is a certain effect on the occurrence of it.
4. the results of Meta analysis also indicated the negative effects of KIR2DL2 on CHC treatment.
5. NK92 cells treated by 5-Aza-dc could express the KIR2DL2 molecules on the surface of the cells. After using the antibody concentration 20 g/ml to seal the KIR2DL2 receptor, the cytotoxicity of NK cells to K562 cells was significantly enhanced.
conclusion
1.KIR2DL2 can inhibit the activity of NK cells.
2. KIR2DL2 and its receptor HLA-C Groupl may be beneficial to the occurrence of CHC in Chinese Han population and the poor response to combined treatment of PeglFN-a and Leigh Bhave Lin.
3. the frequency of KIR2DS3 in CHC patients in Chinese Han population was higher than that in healthy controls. This may be a risk factor for CHC in Chinese Han population.
The frequency of 4. HLA-Cw*06, HLA-Cw*15 and KIR2DS4CHC patients was lower than that of healthy controls. The presence of these genes may inhibit the occurrence of CHC.
The innovation of this study
1. currently, there is a report on the correlation between KIR and CHC in the Caucasus, but there is no related report in the Chinese Han population. This study mainly uses PCR-SSP to detect the correlation between the frequency of KIR and the disease and the treatment of the KIR in the Chinese Han population of the Han population.
2. through the results of the study and Meta analysis, it is the first time to suggest that KIR2DL2 is a negative factor in CHC occurrence and treatment in Chinese Han population.
The significance of this study
1. through the study, it is found that the existence of KIR2DL2 in Chinese Han population has strong NK cell inhibition ability, which makes the existence of KIR2DL2 detrimental to the clearance of HCV, which leads to the poor therapeutic effect of HCV infection, and thus is beneficial to the persistent infection of HCV.
2. K1R2DL2 can be a prejudging index for the effect of HCV treatment, and it is also an epidemiological risk factor for the occurrence of CHC in Chinese Han population. In the later study, KIR2DL2 can become an important target for the immunotherapy of CHC.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.63

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