中西医结合治疗再生障碍性贫血用药选择预测系统的初步研究

【作者】 唐旭东；

【导师】 麻柔；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2008， 博士

【摘要】 目的:研究和评价不同分型再生障碍性贫血(aplastic anemia,AA)患者外周血T细胞亚群、Th1/Th2、T-bet和GATA-3基因表达的变化规律,探讨AA患者不同临床分型、病程以及综合免疫学、分子生物学指标和临床信息等多变量指标的预测系统对AA患者治疗策略选择(免疫抑制剂和雄激素)的预测价值。方法:采用流式细胞术检测85例AA患者治疗前后外周血T细胞亚群及其他免疫学指标,同时运用实时监测定量PCR技术检测T-bet和GATA-3基因,从中医辨证(肾阳虚/肾阴虚)、西医诊断(急性再障/慢性再障)、疗效(缓解/进步/无效)和治疗方法(雄激素有效/无效和免疫抑制剂有效/无效)四个角度对AA患者进行不同分组,寻找表达显著的免疫学和分子生物学指标,筛选有临床意义并有显著差异性的指标,结合临床诊断依据,以雄激素和免疫抑制治疗有效病例做Logistic回归方程及ROC曲线分析,建立以临床和实验指标为主组成的预测系统,对临床治疗用药选择进行预测。结果:1.总计85例AA患者经过两个疗程治疗,总有效率76.5%,其中肾阳虚型和肾阴虚型总有效率分别为97.2%和61.2%,急性再障(acute aplastic anemia,AAA)和慢性再障(chronic aplastic anemia,CAA)的总有效率分别为65.3%和91.7%,肾阳虚型AAA和CAA的有效率分别为100%和96.4%,肾阴虚型AAA和CAA的有效率分别为57.5%和75%,表明肾阳虚型疗效明显高于肾阴虚型,CAA疗效明显高于AAA患者,结果说明中西医分型原则的综合运用有助于预测AA的临床疗效。2.雄激素治疗有效病例以CAA20例(62.5%)、肾阳虚型21例(65.6%)为多;免疫抑制剂治疗有效病例以AAA16例(59.3%)、肾阴虚型17例(63%)为多。由以上可以大致看出,阳虚型和CAA较适宜于用雄激素治疗而AAA和阴虚型较适宜于用免疫抑制剂治疗,但判别准确率仅50%～60%。3.治疗前肾阴虚组外周血象(白细胞、血红蛋白、血小板)较。肾阳虚组低(P＜0.01),补肾中药治疗后肾阳虚组疗效(外周血象恢复)优于肾阴虚组(P＜0.01)。4.肾阴虚组治疗前症状较肾阳虚组重(P＜0.05),治疗后肾阳虚组疗效(症状减轻或消失)明显优于肾阴虚组(P＜0.05)。肾阴虚和肾阳虚组腰脊酸痛和胫酸膝软或足跟痛的频次都较多,说明腰脊酸痛和胫酸膝软或足跟痛是AA肾虚辨证主要症状。中医辨证肾阳虚型患者的临床症状轻,预后好;肾阴虚型患者临床表现重,预后相对较差。5.与对照组相比,治疗前AA患者出现一系列免疫指标变化,治疗后肾阳虚组各免疫指标与正常对照组比较已无差异,而肾阴虚组的免疫功能较肾阳虚组较难恢复;治疗后CAA各免疫指标与正常对照组比较已无差异,AAA的免疫功能较CAA更难恢复。6.分子生物学指标中,研究发现治疗前AA各组患者T-bet/β-actin和T-bet/GATA-3的对数明显低于健康对照组,经补肾中药治疗后恢复,GATA-3/β-actin的对数无显著变化。另从治疗效果分析,T-bet/β-actin的对数治疗前后在缓解组(疗效)都最高(P=0.033,P=0.017),T-bet/GATA-3的对数治疗前在缓解组(疗效)和免疫抑制剂组(治疗方法)最高(P=0.010,P=0.010),治疗后恢复正常;结果提示T-bet/GATA-3可能对雄激素和免疫抑制剂的使用具有预测作用。7.经过判别分析和回归分析,筛选出1g(T-bet/β-actin)、1g(T-bet/GATA-3)、Th1/Th2、CD3~+CD8~+(%)和CD3~+HLA-DR~+(%)共5项最有意义的指标做为预测指标,综合临床诊断标准,得出免疫抑制剂或雄激素选择用药的预测回归方程。8.按SPSS的ROC曲线模型,设定假阳性率为10%,取P值为0.832。若P值大于或等于0.832时,患者判定为免疫抑制为主状态,应以补肾中药加免疫抑制剂为主治疗;若P值小于0.832时,患者判定为骨髓衰竭为主状态,应以补肾中药加雄激素治疗。9.预测方程的临床可行性检验与评价说明,42.2%(14/33)的患者可能受益于预测结果而提高治疗效果。所以,预测方程可能提高临床治疗AA的治疗效率,协助临床医师进行合适的用药选择。结论:1.中医辨证肾阳虚型AA临床症状明显轻于肾阴虚型,中医辨证肾阳虚型AA的临床疗效优于肾阴虚型;2.提出并证明再障的发病大致可分为以异常免疫为主和以骨髓衰竭为主2个阶段,前一阶段以免疫抑制剂+中药活血解毒补肾为主治疗;后一阶段应以雄激素+补肾中药为主治疗,之间可能存在过渡阶段;3.AA的免疫异常阶段以CD3~+CD8~+、CD3~+CD25~+、CD3~+HLA-DR~+、Th1/Th2等免疫学指标和T-bet、GATA-3等基因表达异常为主要特征;4.首次通过判别分析和回归分析建立以免疫学、分子生物学等现代指标和临床信息等多变量组成的预测系统,并确定以异常免疫为主和以骨髓衰竭为主2个阶段的分界点为P=0.832,以选择治疗策略。5.回顾性临床可行性检验显示预测系统的准确率为88.9%,有可能提高再障的临床疗效。更多还原

【Abstract】 Purpose To do researches on T lymphocyte cell subgroups, T helper cells (Th), T-bet and GATA-3 gene expression regularity in aplastic anemia (AA) patients’ peripheral blood. To evaluate the potential usefulness of a multivariable model in predicting the selection of immunosuppressive therapy (IST) or androgens in patients with AA, which coagulate immunology, microbiology and clinical parameters in all, and its application to the clinical practice.Methods 85 patients were selected and treated in general. There were 4 forms of groups to be made according to symptoms of differenciation in traditional Chinese medicine (TCM), diagnosis in modern medicine, effectiveness and therapeutic methods. Peripheral blood T cell sub-groups and Th were serially analyzed by flow cytometer before and after the treatment. At the same time T-bet and GATA-3 genes were also assessed by real time RT-PCR. Then Logistic formulate and ROC curves were made based on cases responding to IST or Androgens. The multivariable model is set up to predict the selection of drugs (IST or Androgens) in clinical practice.Results 1. After treatments for 2 courses, the total effectiveness ratio in kidney-yin deficiency group and kidney-yang deficiency group were respectively 97.2% and 61.2%. In acute aplastic anemia (AAA) and chronic aplastic anemia (CAA) groups they were 65.3% and 91.7% respectively, AAA and CAA in kidney-yang deficiency group were 100% and 96.4%, AAA and CAA in kidney-yin deficiency group were 57.5% and 75%. The above results showed that curative effect in kidney-yang group was higher than kidney-yin group, and CAA higher than AAA group, which administrated that Chinese and western cooperational dignostic methods were helpful to predict therapeutic effect. 2. The curative patients with androgens (62.5%) were more in CAA (62.5%) and kidney-yang deficiency group (65.6%). The therapeutic patients with IST were more in AAA (59.3%) and kidney-yin deficiency group (63%). So from the above data, we could conclude that CAA in kidney-yang deficiency group was better to use androgens and AAA in kidney-yin group was better with IST. However, the accurative ratio was 50%~60%. 3. The peripheral blood in kidney-yin deficiency group was much lower than kidney-yang deficiency group as white blood cell (WBC), hemoglobin (HB) and platelet (PUT) was discussed (P<0.01). And after treatment, the therapeutic effect in kidney-yang deficiency group was better (P<0.01).4. The symptoms in kidney-yin group were more serious than those of kidney-yang deficiency group (P<0.05). The symptoms in kidney-yang deficiency group dicreased and patients recover more obviously (P<0.05). In all the symptoms observed, lumbar ache and painful heel were more often seen and can be made main symptoms of kidney deficiency syndrome. As curation was concerned, kidney-yang deficiency group was more curable than kidney-yin deficiency group. 5. Compared with controls, before the treatment, there were a series of immunological factors differenciation in AA patients. They returned to normal and had no significent differences in kidney-yang deficiency group as with controls, though in kidney-yin group the immune function was not yet recovered and rather contractory. And in diagnostic groups, immunological terms in AAA were more difficult to ameliorate than those in CAA group. 6.The logarithm of T-bet/β-actin in Kidney-Yang group, chronic AA group, partly cured group decreased obviously (P<0.05). After Bushen Chinese herbs’ treatment, The logarithm of T-bet/β-actin changed and lowered in kidney-yin deficiency group, acute AA group and partly cured group (P<0.05). The logarithm of T-bet/GATA-3 in kidney-yin deficiency group, chronic AA group, group of no respondence minimized statistically, which changed to normal level after Chinese herbs’ treatment. The results inferred that T-bet/GATA-3 might have predictions of androgens and IST selections. 7.With the discriminant and logistic statistics, lg(T-bet/β-actin), lg(T-bet/GATA-3), Thl/Th2, CD3~+CD8~+(%) and CD3~+HLA-DR~+(%) 5 indexes were screened out to act as predicting factors. Together with clinical symptoms and diagnosis, the logistic formula was made to predict the selection of IST or androgen drugs. 8. A point between the 2 stages was found out through the calculation of the logistic formulate and ROC curve. The point was 0.832. When P≥0.832, it was in the abnormal immunological stage, IST should be used. When P<0.832, then it was in the bone marrow failure stage, androgens should be added. 9.The predictive formula indications of clinical probabilities and evaluations showed that 42.2%(14/33) patients could have benefits from the predictions made by the formula, which could advance the curative ratio and fanal results. All in all, the predictive system could obviously enhance AA curativeness and did well to clinical physicians in selection of androgens or IST properly and accurately.Conclusions 1. Bushen Chinese herbs have effectiveness and accuracy of treating AA with no side effect. The therapeutic effect of Bushen Chinese herbs is more effeictive in kidney-yang group than in that in kidney-yin group. 2. A noval theory is proposed that there are 2 stages that are abnormal immunological stage and bone marrow failure stage in the course of AA. In the former stage, IST should be used. In the later one, androgens are prefered. 3. Immunologicl factors as CD3~+CD8~+, CD3~+CD25~+, CD3~+HLA-DR~+, Thl/Th2 and microbiological factors as T-bet and GATA-3 genes are the characteristics of abnormal state in AA patients. 4. A formula incorporating laboratory terms and clinical evidence is made to predict selections of IST and androgens. A point of P=0.832 is ascertained as the borderline of 2 states in AA, which are abmoral immunological state and bone marrow failure state. 5. Retrospective clinical probability analysis shows that the exactivity of the prodicting system is 88.9%, probably raising the effectiveness of AA.更多还原