摘要
血小板减少性紫癜原名为“特发性血小板减少性紫癜(Idiopathic thrombocytopenic purpura, ITP)",是一种由于患者体内产生抗自身血小板抗体引起血小板破坏增多,从而导致外周血中血小板持续减少,骨髓巨核细胞数正常或增多伴有成熟障碍为特征的出血性疾病。
本研究目的在于探讨CITP中医辨病辨证治疗思路与方案,并验证参桂益气温阳汤治疗CITP的临床疗效,补充和丰富CITP中医治疗思想,为CITP中医系统化治疗提供参考。探讨CITP患者排除激素干扰下免疫功能紊乱状态。结合T-bet/GATA-3、Foxp3 mRNA转录等分子生物学研究,进一步阐明疗效机制。
一、临床疗效研究
本研究结果显示,治疗组总病例数为28例,显效4例(14.29%)；良效3例(10.74%)；进步5例(17.86%)；无效16例(57.14%)；总有效12例；总有效率42.86%。治疗前中位Plt计数23.02±13.69；治疗后中位计数46.18±50.47,治疗前后血小板变化具有统计意义(P<0.05)。说明参桂益气温阳汤一定程度提高外周血小板水平。后期观察6个月,显效病例中1例4个月后plt降至80×109／L,目前波动在80×109/L-90×109/L,余病例病情稳定。全部病例观察期间未出现不适不良反应。
二、参桂益气温阳汤疗效机理研究
治疗组符合入组条件CITP病例共25例,男：女=1：1.5,中位年龄35.82±17.70。健康空白对照20例,男：女=1：1.14,中位年龄33.45±15.42。两组性别比例及中位年龄无明显差异(P>0.05)。
利用美国BECKMAN COULTER公司流式细胞仪检测CD3+T淋巴细胞、CD3+CD4+T淋巴细胞、CD3+CD8+T淋巴细胞、CD4+／CD8+比例、CD3+D25+活化T细胞、CD3+DR+活化T细胞、CD4+CD25+Treg细胞、NKT细胞、NK细胞、CD19+B淋巴细胞、CD19+CD5+B淋巴细胞及Th1/Th2/Th0治疗前后表达变化。
此次淋巴细胞亚群研究提示,CITP患者机体免疫紊乱中Treg细胞和CD19+CD5+B细胞比例下调及功能缺陷、以及总T细胞、CTL细胞、NKT细胞、CD3DR+活化T细胞的功能亢进是其致病机制中重要环节。治疗前后研究提示,参桂益气温阳汤通过显著降低总T细胞、NKT细胞、CD3DR+活化T细胞比值；升高CD19+CD5+／CD19+比值、CD4+CD25+Treg细胞和Th2细胞,起到免疫调节和免疫耐受作用,有助于改善外周血小板水平。值得注意的是,治疗无效组CTL细胞和CD3+DR+活化T细胞高表达,并不因治疗干预而下降,说明CTL细胞和CD3+DR+活化T细胞功能增强可能与外周血小板持续破坏有关。
经SPSS statistics 17.0进行多元逐步回归分析,提示治疗前后各项T、B淋巴细胞亚群与PBC之间无线性相关性。
三、转录因子T-bet、GATA-3、Foxp3mRNA研究
利用实时定量逆转录PCR检测治疗前后转录因子T-bet、GATA-3、Foxp3mRNA转录水平,探讨与Th1、Th2、CD4+CD25+Treg细胞以及PBC间相关性。
结果显示：治疗组治疗前GATA-3mRNA转录水平显著高于健康对照组,差异具有统计学意义(P＜0.01),经治疗表达水平显著下降,治疗前后变化具有显著性差异(P<0.01),治疗后与健康对照无统计学差异。Foxp3mRNA转录水平治疗前与健康对照无差异,治疗后Foxp3mRNA表达显著低于健康对照,具有显著性差异(P＜0.05)。
治疗前GATA-3mRNA转录水平增强考虑与机体的基因层面自身调控有关,通过GATA-3mRNA高表达,使Th平衡向Th2优势漂移,有利于免疫耐受和病情缓解,且随着治疗反应和Treg调节细胞的上升而表达逐渐下降,符合疾病的缓解过程。Foxp3mRNA转录水平经治疗有所下降,说明随着疾病缓解外周Treg调节细胞比例显著上升和功能增强,负反馈地调节基因转录水平,使其表达下调。证明Foxp3虽然是Treg特异性调控因子,但也接受Treg的负反馈调节,这是机体动态平衡奥妙所在。
运用SPSS statistics17.0统计软件对治疗前后转录因子T-bet与Th1、GATA-3与Th2、Foxp3与Treg细胞进行双变量相关分析,结果显示各转录因子与调控靶细胞之间无线性相关性(P＞0.05)。
运用SPSS统计软件对治疗前后转录因子T-bet、GATA-3、Foxp3三因素间进行双变量相关分析。结果显示：治疗前,T-bet与GATA-3呈负相关(P＜0.05),但相关性不甚密切(相关系数<0.5)；与Foxp3呈显著负相关(P＜0.01,相关系数>0.5)。治疗后,Foxp3与GATA-3之间呈现正相关关系(P<0.05),相关系数为0.427。提示CITP慢性期Foxp3、GATA-3和T-bet之间存在相互制约关系,这符合CITP疾病状态及各转录因子的功能。缓解期Foxp3和GATA-3转录相伴降低,说明随着外周Treg调节细胞的恢复和功能增强,通过负反馈机制降低了Foxp3和GATA-3转录水平。
运用SPSS statistics17.0多元逐步回归分析进一步探讨转录因子T-bet、GATA-3、Foxp3表达与PBC之间相关性。结果显示,治疗后GATA-3与PBC呈负相关,P=0.032(P＜0.05),得出方程式plt=90.299-64.086gata。说明疾病缓解,外周血小板上升与GATA-3转录水平下降有一定相关性。
本研究尚存在不足之处。一、转录因子及淋巴细胞亚群为敏感性实验指标,随着疾病状态变化而波动,本实验观察期4-5个月,只是抽取治疗前后血样作为研究指标,有可能丢失了动态变化过程,不利于全面客观地阐述致病机理。本研究不排除错过了捕捉和研究转录因子动态变化的机会。二、治疗有效病例数有限,相对影响了疗效机理研究的客观性,难免存在统计误差。
本研究提示CITP疾病未缓解状态与CD3+CD8+CTL细胞和CD3+R+活化T细胞高表达密切相关。据此设想今后研究中加强CTL细胞、活化T细胞的研究深度,随着颗粒酶、穿孔素,IL-2、IFN-γ的血浆浓度变化和相关基因调控机理的明晰,将有助于进一步阐明CITP致病机理。
Thrombocytopenic purpura was named Idiopathic thrombocytopenic purpura(ITP) at the beginning. In 2007 the International Working Group(IWG) recommended to change its name to Immune thrombocytopenia(ITP). The word "Immune", was to emphasize that the cause of the disease was immune, In addition, some patients have no or have light hemorrhage, so the word "purpura" should be abandoned, named "immune thrombocytopenia". However, its abbreviation retains "ITP".
Based on his many years temporary experience, professor Ma Rou, my tutor, proposed a series of therapeutic regimen that committed not only to the disease but also to the patients symptoms. Professor Ma Rou subtyped CITP into two groups: spleen-kidney yang deficiency and dual deficiency of the lung-spleen. He used the method of tonifying qi and warming yang, and set up Shenguiyiqiwenyang decoction to therapy CITP and obtained good effects.
The Objective of this study was to discuss tutor's therapeutic ways for CITP and verify its clinical effect, and to discuss the immune disorderly conditions of CITP's patients without glucocorticoid interfering. Together with T-bet/GATA-3,Foxp3 mRNA transcribe molecular biology study, further expounded the therapeutic mechanisms of Shenguiyiqiwenyang decoction.
The results was that in treatment group(28 cases),4 cases(14.29%) were excellence, 3 cases(10.74%) were good,5 cases(17.86%) were improvement,16cases(57.14%) were invalid. That is,12cases were effective and total effective rate was 42.86%.
The platelet counts was 23.02±13.69 before treatment in treatment group, and after treatment was 46.18±50.47.And there was significant difference(P<0.05)between before and after treatment. It proveed that Shenguiyiqiwenyang formula had significant curative effect for ITP.
We used flow cytometry of BECKMAN COULTER to detect CD3+T lymphocyte, CD3+CD4+T lymphocyte, CD3+CD8+T lymphocyte, CD4+/CD8+proportion, CD3+CD25+ active T lymphocyte, CD3+DR+ activate T lymphocyte, CD4+CD25+ Treg, CD3+CD16CD56+ NKT cell, CD3-CD16CD56+NK cell, CD19+B lymphocyte, CD19+CD5+ B lymphocyte and the change of Thl/Th2/ThO between before and after treatment.
Compared to the healthy group, they increased significantly(P<0.05) with CD3+CD19- Total T lymphocyte, CD3+CD8+T lymphocyte, NKT cell, CD3DR+ active T lymphocyte cell counts, and decreased significantly(P<0.05) with CD4+/CD8+ proportion, CD19+CD5+/CD19+ cell proportion, CD4+CD25+Treg cell counts of before treatment in treatment group. And after treatment,they decreased significantly(P<0.05) with CD3+CD19- Total T lymphocyte, NKT cell, CD3DR+ active T lymphocyte cell counts, and increased very significantly(P<0.01) with CD19+CD5+/CD19+ cell proportion, CD4+CD25+Treg cell counts. There was no significant difference of CD3+CD8+T lymphocyte,CD4+/CD8+ proportion and Th1、Th2、Th0 between before and after treatment in treatment group.
In effect group, the result showed that:CD4+CD25+Treg cell proportion increased significantly(P<0.05) after treatment. In inefficacy group, the study showed that: after treatment, CD3+CD19- Total T lymphocyte proportion decreased significantly(P<0.05); CD4+CD25+Treg cell counts CD19+CD5+/CD19+ cell proportion increased significantly(P<0.05). Compared to before treatment there was no significant difference with CD3+CD8+ CTL cell and CD3DR+ cell after treatment, but compared to healthy control group, there was significant difference(P<0.05).
In all, in CITP patient organism, we can deduce that the low CD4+CD25+Treg cell and CD19+CD5+B cell expressed low level and had immunologic function defect and CD3+CD19- Total T lymphocyte CD3+CD8+ CTL cell NKT cell and CD3DR+ cell had hyperfunction, All that were important link in pathogenic mechanism. The target improved after treatment, which evident further that factor had important effect in pathogenesy. It's worth noting that in inefficacy group CD3+CD8+ CTL and CD3DR+ cell expressed high level, and had not descend with therapy interfering in. What demonstrateed:CTL cell and CD3DR+ active cell were important factor in CITP disease course.
By multivariantly gradual regressive analysis, there were no linear correlations among every T,B lymphocyte subset and peripheral blood cells.
Using Real-Time-RT-PCR, we detected transcription factor T-bet, GATA-3, Foxp3 mRNA transcriptional level in before treatment and after treatment, and discussed associativity among transcription factor's transcriptional level, Th1, Th2, CD4+CD25+ cell and peripheral blood cells.
The results showed that:in the treatment group, GATA-3mRNA transcriptional level was notably higher than in healthy control group(P<0.01). After treatment, the transcriptional level decreased significantly(P<0.01). There had no statistical significant difference comparing after treatment in treatment group with healthy control group. T-bet mRNA transcriptional level had no statistical significance comparing before and after treatment in treatment group with healthy control group. The transcriptional level of Foxp3mRNA had no statistical significance comparing before treatment in treatment group with healthy control group, but it decreased in after treatment. After treatment, the transcriptional level of Foxp3mRNA was lower than healthy control group(P<0.05).
Before treatment, GATA-3mRNA transcriptional level was strengthened, which concerned with autoregulation reaction in body's gene deck. The high expressing of GATA-3mRNA made Th balance drift toward Th2 superiority, that profited to immune tolerance and patient's condition relieving. With therapeutic reaction and Treg accommodate cell's raising, GATA-3 mRNA transcriptional level descended. It consistent with the disease variation processed. Foxp3mRNA transcriptional level descended after treatment, this indicated that peripheral Treg accommodate cell population increased and it's function strengthened with the disease remission and then adjusted genetic transcription level by negative feedback, making it's level express down. It proved that although Foxp3 was the Treg's specific regulating factor, which accepted Treg's negative feedback regulation.
Using the SPSS 17.0, we analyzed the double variable relations among the 3 factors of transcription factors of T-bet、GATA-3、Foxp3 of before and after treatment. Before treatment, T-bet and GATA-3 was negative correlation ship(P<0.05), but dependability was not very intimate(coefficient correlation<0.5); with Foxp3 was in obviously negative correlation(P<0.01, coefficient correlation>0.5). After treatment, Foxp3 and GATA-3 was in positive coefficient correlation(P<0.05), coefficient correlation was 0.427.It proved that GATA-3 feedback high expression will restrain relatively T-bet transcription in CITP chronic phase; Foxp3 transcription in high level will restrain T-bet function too, which was in line with the morbid state of CITP and in line with functions of every transcription factor. In paracmasis, the transcription of Foxp3 and GATA-3 degrade concomitantly, which proved that along with the regaining of the periphery blood Treg adjusting cell and the improving of the immune transient status, the level of transcription about Foxp3 and GATA-3 was be degraded by degenerative feedback mechanism.
By multiple linear regression analysis, we further investigated the correlation among transcription factor T-bet、GATA-3、Foxp3 expression and peripheral blood cells. The result showed, after treatment GATA-3 and PBC was negative correlation(P<0.05), and the equation was PLT=90.299-64.086gata. It descripted that the ascensus of platelet in peripheral blood had dependability with the descending of GATA-3 transcriptional level.
To summarize above all, through the study, we can further verified that Shenguishenqiwenyang decoction is effective and safe for CITP patiets, and its prostecdtive efficacy (further observing 6 months after platelet ascensus) is stable. The research of mechanism detected that the proportion of CD4+CD25+Treg cells increased notably(P<0.05) in utility groups(excellence+good). In ineffective group, CD3+CD19-Total T lymphocyte proportion decreased after treatment, CD4+CD25+ Treg cell counts CD19+CD5+/CD19+ cell proportion increased, and there was significant difference(P<0.05); CD3+CD8+ CTL cell and CD3DR+ cell keep still high expression, they descend rarely after treatment(P<0.05). The study indicated that it was the possible remission mechanism that CD4+CD25+Treg cell and CD19+CD5+/CD19+ cell proportion increased or their function strengthened, Meanwhile,CD3+CD8+ CTL cell NKT cell and CD3DR+ active T cell functions restrained. It's worth noting that in ineffective group CD3+CD8+ CTL and CD3DR+ cell expressed with high level, and they did not descend with therapy, so this demonstrate that CTL cells and CD3DR+ active cells were the important factor in CITP disease course.
引文
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