重型肝炎与HBV PreS区基因突变的关系研究及中医干预的疗效观察
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摘要
重型肝炎是由多种原因引起的急性大量肝细胞坏死和严重肝功能障碍以致肝功能突然减退或消失,迅速出现凝血功能障碍、肝性脑病、肾功能损害和多脏器功能衰竭的一种临床危急重症。在我国,乙型肝炎病毒(hepatitis B virus, HBV)感染所致的慢性乙型重型肝炎(chronic severe hepatitis B, CSHB)约占全部慢性重型肝炎90.29%,临床证候复杂,变证兼夹证多,治疗难度大,预后不良,存活者病情仍可多次反复,病死率高达60%-80%,近年来采用中西医结合治疗取得一定疗效,使病死率有所下降。
既往研究提示HBV基因型、HBV核苷酸突变可能和乙型肝炎患者的病情加重有关,但与CSHB之间的关系仍还不明确,少数研究提示可能和PreS区的基因突变缺失有一定的关系,但目前也未有定论。
有鉴于此,本文进行了以下研究：①临床研究,观察中西医结合治疗CSHB患者的临床疗效；②实验研究,探讨HBV基因型、PreS区基因突变与CSHB发生之间的关系。
第一部分临床研究
目的：
观察中西医结合治疗CSHB患者的临床疗效。
方法：
选取2010年3至2011年8月在广州市第八人民医院住院的CSHB患者36例,随机分为治疗组19例和对照组17例。治疗组采用中西医结合治疗措施,对照组给予单纯西医治疗。比较两组患者治疗4周后的主要实验室指标、并发症发生率、中医临床症状积分和临床疗效。比较48周随访结束时两组的死亡率,分析CSHB患者死亡的相关因素。
结果：
(1)在肝功能指标方面：治疗组的总胆红素(total bilirubin, TBIL)和凝血酶原活动度(prothrombin time activity, PTA)在治疗后均得到显著改善,治疗前后自身比较,差异有统计学意义(P<0.05),而对照组中均无明显区别(P>0.05)；治疗组在降低TBIL上显著优于对照组(P<0.05)。两组的谷丙转氨酶(alanine aminotransferase, ALT)在治疗后显著下降、白蛋白(albumin, ALB)明显升高,治疗前后组内比较,差异具有统计学意义(P<0.01或P<0.05),组间比较差异不明显(P>0.05)。治疗后两组的总胆汁酸(total bile acid, TBA)和胆碱酯酶(cholinesterase, CHE)无明显改善,治疗前后组间、组内比较,均无统计学差异(P>0.05)。
(2)在肾功能指标方面：两组患者的尿素氮(blood urea nitrogen, BUN)和肌酐(creatinine, Cr)水平在治疗前后均处于正常范围内,治疗前后组间、组内比较,差异均无统计学意义(P<0.05)。
(3)在血氨(blood ammonia. BLA)水平方面：两组治疗前后无发生明显变化,组间、组内比较均无统计学差异(P>0.05)。
(4)在血小板(platelets, PLT)水平方面：治疗组的下降幅度显著小于对照组(P<0.05)；治疗前后组内比较,治疗组差异不明显(P>0.05),而对照组差异显著,具有统计学意义(P<0.01)。
(5)在HBV DNA水平方面：治疗组在治疗后HBV DNA转阴率为10.5%(2/19),对照组为17.6%(3/17),两组比较差异无统计学意义(P>0.05)。两组的HBV DNA水平较治疗前均出现显著下降,组内比较,差异均有统计学意义(P<0.01)；组间比较,无统计学差异(P>0.05)。
(6)在并发症发生率方面：治疗组在降低原发性腹膜炎发生率及预防真菌感染上,优于对照组(P<0.05和P=0.09)。
(7)在中医症状积分方面：治疗后两组均得到改善,治疗前后组内比较,治疗组差异显著(P<0.01),而对照组无统计学差异(P>0.05)。治疗组在降低中医临床症状积分方面显著优于对照组(P<0.05)。
(8)在临床疗效方面：经治疗后,治疗组的总有效率和显效率分别为73.7%(14/19)和47.4%(9/19),对照组分别为41.2%(7/17)和11.8%(2/17),治疗组的综合疗效及显效率均明显优于对照组(P<0.05)。
(9)在病死率方面：在48周随访结束时,治疗组病死率为21.1%(4/19),低于对照组的41.2%(7/17),但两组间差异无统计学意义(P>0.05)。
(10)在死亡相关因素方面：死亡组的甲胎蛋白(alpha-fetoprotein, AFP)水平显著偏低(P<0.01),Cr水平及中医临床症状积分明显偏高(P<0.05)。死亡组在所有并发症的发生率上均高于存活组,在真菌感染、原发性腹膜炎、肝性脑病的发生率上与存活组相比,具有显著性差异(P<0.01)。
结论：
(1)对CSHB患者采用中西医结合治疗,可以促进患者黄疸消退,减缓PLT下降速度,改善患者的临床症状,降低中医症状积分及并发症的发生率,提高临床疗效,降低病死率。
(2) CSHB患者在基线时AFP水平越高则其预后相对越好；而治疗期间并发症发生率越高则预后相对越差。
第二部分实验研究
目的：
探讨HBV基因型、PreS区基因突变与CSHB发生之间的关系,寻找与CSHB可能相关的特异性核苷酸突变位点。
方法：
选取2010年3月至2011年8月广州市第八人民医院住院的120例乙肝病毒感染者,其中慢性乙型肝炎患者(choronic hepatitis B, CHB)43例、肝硬化患者(liver cirrhosis, LC)21例、肝细胞癌患者(hepatocellular carcinoma, HCC)20例、CSHB患者36例(同第一部分研究病例)。共采集到血清样本232份,其中CSHB患者在治疗前后及随访期共采集到148份。采用巢式PCR法,检测HBV基因型,扩增HBV PreS基因并进行DNA测序。通过对比分析所有血清样本的HBV基因型分布、PreS区基因突变位点的发生率,探讨HBV基因型、PreS区基因突变与CSHB发生的关系。
结果：
(1)HBV基因型研究
1)CHB组、LC组、HCC组、CSHB组的HBV基因型分布的组间差异显著,有统计学意义(P<0.05), CHB组、CSHB组以B基因型为主,LC组、HCC组以C基因型为主,进一步两两比较显示：CSHB组与LC组、CSHB组与HCC组差异显著,均有统计学意义(P<0.01)。
2)B基因型与C基因型间的临床资料比较：CHB组中各指标在两基因型间的差异不明显,均无统计学意义(P>0.05)；LC组中,C基因型中的HBV DNA和PLT水平高于B基因型(P<0.05和P=0.071); HCC组中,C基因型中的PLT水平更高、Cr水平更低,两基因型间差异明显,均有统计学意义(P<0.05)；CSHB组中,C基因型中的HBeAg阳性率更高,TBA、ALT、ALB、CHE水平偏低,差异较为显著,但均无统计学意义(P值依次为：0.071、0.064、0.054、0.055和0.093)。
3)基因B型与基因C型在CSHB患者的临床分期、临床疗效、死亡率上比较,差异均无统计学意义(P>0.05)。
4) CSHB组的HBV基因型纵向对比显示：9例临床治愈的患者中有2例发生基因型B→B/C→C型的改变。
(2) HBV PreS区基因突变研究
1)成功扩增PreS区基因片段的62份血清样本中,61份均发生了点突变,检出率在前5位的突变位点分别是：A2873(93.5%)、T3041(91.9%)、G3150(93.5%)、 A46(93.5%)、A49(64.5%)。四组患者的PreS区突变位点总数为：HCC组>CSHB组>LC组>CHB组,四组间的差异显著有统计学意义(P<0.01)。组间两两比较：LC组与CSHB组比较无统计学差异(P>0.05),其余均存在显著性差异(P＜0.01)。
2)比较CSHB组与CHB组PreS区基因突变：A2875、C2901、C2910、C2922、A2951、G2962、A2964、C2980、 G2988、G3006、G3021、C3057、T3060、G3063、 A3066、G3069、C3097、G3102、T3108、G3156、T3169、A3171、G3186、C3191、 A20、T25、A27、C40、A76、G85、G87、C93、A96、A99、T105、G110、A127、 A132、T147共39个位点的突变率有统计学差异(P<0.01或P<0.05),OR值均大于1。
3)比较CSHB组与LC组PreS区基因突变：A2946、C3009位点突变率有统计学差异(P<0.05),OR值小于1。
4)比较CSHB组与HCC组PreS区基因突变：C3009、G3156位点突变率有统计学差异(P<0.05),OR值小于1。
5) PreS1区和PreS2区起始密码子缺失和突变分析：CSHB组中：PreS1区2例(8.3%,2/24), PreS2区8例(33.3%,8/24)；CHB组中：PreSl区、PreS2区各1例(4.2%,1/24)；LC组中：PreS2区2例(20%,2/10)；HCC组中：PreS2区3例(75%,3/4)。四组患者均以PreS2区起始密码子缺失突变为主,CSHB组、HCC组的突变率都明显高于CHB组(前者P<0.05,后者P<0.01),OR值均大于1。
6) CSHB死亡组与存活组在PreS区基因突变数及点突变率上比较,均无统计学差异(P>0.05)。
7) CSHB组的PreS区基因突变纵向对比显示：3例CSHB患者在治疗前后突变位点数发生明显改变,CSHB-6:0周11个→4周70个；CSHB-9:0周10个→4周64个→24周9个；CSHB-15:0周69个→12周11个。
结论：
(1) CSHB患者的HBV基因型以B基因型为主,B基因型与C基因型的疾病严重程度相似,不同基因型对患者的临床疗效及最终结局的影响无明显区别。
(2)在治疗过程中,9例临床治愈的CSHB患者中,有2例出现HBV基因型由B→B/C→C的改变,基因型改变无加重患者病情。
(3) CSHB组的PreS区基因突变数及多个位点的突变率明显高于CHB组,PreS区变异与CSHB发生密切相关。
(4) CSHB患者的PreS区基因突变率在治疗期间可呈动态变化,在疾病进展期高,随着病情的好转,突变率下降,突变位点逐渐恢复。
Severe hepatitis is a kind of acute clinical critical disease caused by a variety of reasons with acute necrosis of massive hepatic cells and severe liver dysfunction so that liver function suddenly reduce or disappear, coagulation disorders, clinical manifestations as hepatic encephalopathy, renal damage and multiple organ failure. In China,90.29%of severe hepatitis is chronic severe hepatitis B (CSHB), with complicated clinical syndromes, variable complications, difficulty in treatment, high mortality rate, poor prognosis, continuous onsets in survivors, and the fatality was reported about60%to80%. In recent years, we have considerable advantage in researches into integrating therapy of Chinese and Western medicine, and make the death rate decline to a certain extent.
Previous studies suggested that hepatitis B virus (HBV) genotype and HBV nucleotide mutations may be related to exacerbations of hepatitis B hepatitis, but the relationship between the occurrence of CSHB and HBV mutations in patients with chronic hepatitis B is not clear. The existing research suggested that the mutations in PreS area may be involved in occurrence of CSHB, but also not conclusive.
Therefore, we carried out the following studies in this paper. In the clinical research part, clinical efficacy of integrated therapy of Chinese and Western medicine in patients with CSHB was observed. In the experimental research part, the relationship among HBV genotype, gene mutations in PreS area and the occurrence of CSHB was studied.
Part one Clinical research
Objective:
To observe the clinical efficacy of integrated therapy of Chinese and Western medicine for patients with CSHB.
Method:
Thirty-six patients with CSHB from the Eighth People's hospital during March2010to August2011were randomly divided into two groups treated with the integrated therapy of Chinese and Western medicine (treatment group,19cases) or western medicine only (control group,17cases). The main laboratory index, incidence of complications, score of traditional Chinese medical syndromes (TCM score) and clinical efficacy were monitored before and after4weeks treatment. The mortality was observed at the end of the follow-up and the death related factors of CSHB were analyzed.
Results:
(1) The total bilirubin (TBIL) and prothrombin time activity (PTA) of treatment group were significantly improved after treatment compared with that of the pre-treatment (P<0.05). The treatment group show better effect in reducing TBIL compare with the control group (P<0.05). The alanine aminotransferase (ALT) and albumin (ALB) of both groups were obviously improved after treatment compared with that of the pre-treatment (P<0.01or P<0.05), but there was no significantly difference between groups (P>0.05). The total bile acid (TBA) and cholinesterase (CHE) showed no significant change in both groups before and after treatment (P>0.05).
(2) The blood urea nitrogen (BUN) and creatinine (Cr) were in normal range before and after treatment, there was no significant difference within and between groups (P>0.05).
(3) Blood ammonia (BLA) showed no significant differences within and between groups before and after treatment.
(4) Platelets (PLT) was significantly descended in control group (P<0.01), and the difference was significantly compare with treatment group (P<0.05).
(5) After treatment, the negative conversion ratio of HBV DNA in treatment group and control group were10.5%and17.6%, there was no significance variation between groups (P>0.05). The level of HBV DNA was obviously descended, significant difference showed within groups (P<0.01) but not between groups (P>0.05).
(6) The incidences of primary peritonitis and fungal infection were significantly lower in treatment group (P<0.05, P=0.09, successively).
(7) Compare with pre-post treatment, TCM score descended obviously in treatment group (P<0.01) but not in control group (P>0.05). And significant difference showed between two groups in reducing TCM score (P<0.05).
(8) Total effective rate and significant effective rate in treatment group were73.7%
and47.4%respectively, and41.2%and11.8%in the control group, after treatment. The comprehensive curative effect and significant efficiency in treatment group were statistically significant over those of control group (P<0.05).
(9) At the end of the follow-up, the mortality was21.1%in treatment group, and was41.2%in the control group, the difference between groups showed no significance (P>0.05).
(10) Univariate analyses showed that alpha-fetoprotein (AFP), Cr and TCM score contributed statistically to the clinical outcome of patients alive or dead (P<0.01or P<0.05). The death group had higher incidences of all the complications, and in fungal infection, primary peritonitis and hepatic encephalopathy were significantly higher than survival group (P<0.01).
Conclusion:
(1) Integrating therapy of Chinese and Western medicine apply in treatment of patients with CSHB was beneficial to patients with jaundice faded faster and PLT decline slower. The treatment group had better effect on improving clinical symptoms, relieving TCM scores, decreasing complications, improving clinical efficacy and reducing mortality of patients.
(2) Good prognosis in the CSHB patients with higher level of AFP before treatment, while poor prognosis with higher incidence of complications during the treatment.
Part two Experimental research
Objective:
Explore the relationship among HBV genotypes, gene mutations in PreS area and the occurrence of CSHB. And find the possible CSHB-related mutations.
Method:
One hundred and twenty HBV infected patients from the Eighth People's hospital during March2010to August2011were recruited into the present study, including43cases of patients with chronic hepatitis B (CHB),21cases of patients with liver cirrhosis (LC),20cases of patients with hepatocellular carcinoma (HCC), and36cases of patients with CSHB (the same patients in the part one). Totally232serum samples were collected and148serum samples of CSHB patients were collected pre-post treatment and during the follow-up. Nest-PCR was employed for the amplification of the HBV genotype and PreS gene, and PCR product of PreS gene was sequenced. Then the relationship among HBV genotypes, gene mutations of PreS area and the occurrence of CSHB was analyzed.
Results:
(1) Research of HBV genotype
1) The distribution of HBV genotype in CHB group, LC group, HCC group and CSHB group was significant different (P<0.05). The major HBV genotypes in CHB group and CSHB group were genotype B, and genotype C appeared primarily in LC and HCC groups. The distribution of HBV genotypes in CSHB showed significant difference with LC group and HCC group (P<0.01).
2) In CHB group, there were no significant differences in clinical materials of genotype B and genotype C (P>0.05). In LC group, the level of HBV DNA and platelets (PLT) in genotype C were obviously higher than genotype B (P<0.01, P=0.071, successively). In HCC groups, the level of PLT and Cr displayed significantly difference between genotype B and genotype C. In CSHB group, the HBeAg rate was higher and TBA, ALT, ALB, CHE were lower in genotype C (P values in the order:0.071,0.064,0.055and0.093, compare with genotype B).
3) The clinical stages, mortality and clinical effective in CSHB group showed no significant difference between genotype B and genotype C.
4) Longitudinal analysis of genotypes in CSHB group showed that genotype in two cases of nine patients with clinical cure changed from B to B/C then last to C.
(2) Research of gene mutation in HBV PreS area
1) Spot-mutations were found in61of62amplified gene segments of PreS area. Five more mutation sites were A2873(93.5%), T3041(91.9%), G3150(93.5%), A46(93.5%) and A49(64.5%). The mutations of PreS area from most to least was HCC group, CSHB group, LC group and CHB group, the difference being significant (P<0.01). All significant difference was detected between two groups (P<0.01) except between LC group and CSHB group (.P>0.05).
2) Compared CSHB and CHB group mutation showed A2875, C2901, C2910, C2922, A2951, G2962, A2964, C2980, G2988, G3006, G3021, C3057, T3060, G3063, A3066, G3069, C3097, G3102, T3108, G3156, T3169, A3171, G3186, C3191, A20, T25, A27, C40, A76, G85, G87, C93, A96, A99, T105, G110, A127, A132, T147together39units place mutation had the significance (P<0.01or P<0.05), all OR>1.
3) Compared CSHB and LC group mutation showed A2946and C3009had the significance (P<0.05), all OR<1.
4) Compared CSHB and HCC group mutation showed C3009and G3156had the significance (P<0.05), all OR<1.
5) The initiation codon deletion and mutation of PreS1and PreS2were2cases (8.3%) and8cases (33.3%) in CSHB group,1case (4.2%) and1case (4.2%) in CHB group,0case and2cases (20%) in LC group, and0case and3cases (75%) in HCC group. Compared with CHB group, the deletion and mutation rate of PreS2was significant higher in CSHB group (P<0.05) and HCC group (P<0.01), all OR>1.
6) There did not show any significant difference in PreS gene mutation between death group and survival group (P>0.05).
7) Longitudinal analysis of PreS gene mutation in CSHB group showed that the number of mutations obviously changed in three patients before and after treatment. The numbers changed from11(0week) to70(4week), from10(0week) to64(4week) then last to9(24week), and from69(0week) to11(12week) in patient of CSHB-6, CSHB-9and CSHB-15, successively.
Conclusion:
(1) In CSHB patients, genotype B appeared primarily, clinical situation, clinical efficacy, and clinical outcome were similar between genotype B and genotype C.
(2) During the treatment, two cases of nine CSHB patients with clinical cure changed from B to B/C then to C, and this change did not worsen the situation.
(3) Gene mutation of PreS area was possibly related to the occurrence of CSHB and which was significant higher than that in CHB group.
(4) Gene mutations of PreS area changed dynamically in patients with CSHB during the treatment. It appeared more in progression stage, but along with the condition improved, its number decline.
既往研究提示HBV基因型、HBV核苷酸突变可能和乙型肝炎患者的病情加重有关,但与CSHB之间的关系仍还不明确,少数研究提示可能和PreS区的基因突变缺失有一定的关系,但目前也未有定论。
有鉴于此,本文进行了以下研究：①临床研究,观察中西医结合治疗CSHB患者的临床疗效；②实验研究,探讨HBV基因型、PreS区基因突变与CSHB发生之间的关系。
第一部分临床研究
目的：
观察中西医结合治疗CSHB患者的临床疗效。
方法：
选取2010年3至2011年8月在广州市第八人民医院住院的CSHB患者36例,随机分为治疗组19例和对照组17例。治疗组采用中西医结合治疗措施,对照组给予单纯西医治疗。比较两组患者治疗4周后的主要实验室指标、并发症发生率、中医临床症状积分和临床疗效。比较48周随访结束时两组的死亡率,分析CSHB患者死亡的相关因素。
结果：
(1)在肝功能指标方面：治疗组的总胆红素(total bilirubin, TBIL)和凝血酶原活动度(prothrombin time activity, PTA)在治疗后均得到显著改善,治疗前后自身比较,差异有统计学意义(P<0.05),而对照组中均无明显区别(P>0.05)；治疗组在降低TBIL上显著优于对照组(P<0.05)。两组的谷丙转氨酶(alanine aminotransferase, ALT)在治疗后显著下降、白蛋白(albumin, ALB)明显升高,治疗前后组内比较,差异具有统计学意义(P<0.01或P<0.05),组间比较差异不明显(P>0.05)。治疗后两组的总胆汁酸(total bile acid, TBA)和胆碱酯酶(cholinesterase, CHE)无明显改善,治疗前后组间、组内比较,均无统计学差异(P>0.05)。
(2)在肾功能指标方面：两组患者的尿素氮(blood urea nitrogen, BUN)和肌酐(creatinine, Cr)水平在治疗前后均处于正常范围内,治疗前后组间、组内比较,差异均无统计学意义(P<0.05)。
(3)在血氨(blood ammonia. BLA)水平方面：两组治疗前后无发生明显变化,组间、组内比较均无统计学差异(P>0.05)。
(4)在血小板(platelets, PLT)水平方面：治疗组的下降幅度显著小于对照组(P<0.05)；治疗前后组内比较,治疗组差异不明显(P>0.05),而对照组差异显著,具有统计学意义(P<0.01)。
(5)在HBV DNA水平方面：治疗组在治疗后HBV DNA转阴率为10.5%(2/19),对照组为17.6%(3/17),两组比较差异无统计学意义(P>0.05)。两组的HBV DNA水平较治疗前均出现显著下降,组内比较,差异均有统计学意义(P<0.01)；组间比较,无统计学差异(P>0.05)。
(6)在并发症发生率方面：治疗组在降低原发性腹膜炎发生率及预防真菌感染上,优于对照组(P<0.05和P=0.09)。
(7)在中医症状积分方面：治疗后两组均得到改善,治疗前后组内比较,治疗组差异显著(P<0.01),而对照组无统计学差异(P>0.05)。治疗组在降低中医临床症状积分方面显著优于对照组(P<0.05)。
(8)在临床疗效方面：经治疗后,治疗组的总有效率和显效率分别为73.7%(14/19)和47.4%(9/19),对照组分别为41.2%(7/17)和11.8%(2/17),治疗组的综合疗效及显效率均明显优于对照组(P<0.05)。
(9)在病死率方面：在48周随访结束时,治疗组病死率为21.1%(4/19),低于对照组的41.2%(7/17),但两组间差异无统计学意义(P>0.05)。
(10)在死亡相关因素方面：死亡组的甲胎蛋白(alpha-fetoprotein, AFP)水平显著偏低(P<0.01),Cr水平及中医临床症状积分明显偏高(P<0.05)。死亡组在所有并发症的发生率上均高于存活组,在真菌感染、原发性腹膜炎、肝性脑病的发生率上与存活组相比,具有显著性差异(P<0.01)。
结论：
(1)对CSHB患者采用中西医结合治疗,可以促进患者黄疸消退,减缓PLT下降速度,改善患者的临床症状,降低中医症状积分及并发症的发生率,提高临床疗效,降低病死率。
(2) CSHB患者在基线时AFP水平越高则其预后相对越好；而治疗期间并发症发生率越高则预后相对越差。
第二部分实验研究
目的：
探讨HBV基因型、PreS区基因突变与CSHB发生之间的关系,寻找与CSHB可能相关的特异性核苷酸突变位点。
方法：
选取2010年3月至2011年8月广州市第八人民医院住院的120例乙肝病毒感染者,其中慢性乙型肝炎患者(choronic hepatitis B, CHB)43例、肝硬化患者(liver cirrhosis, LC)21例、肝细胞癌患者(hepatocellular carcinoma, HCC)20例、CSHB患者36例(同第一部分研究病例)。共采集到血清样本232份,其中CSHB患者在治疗前后及随访期共采集到148份。采用巢式PCR法,检测HBV基因型,扩增HBV PreS基因并进行DNA测序。通过对比分析所有血清样本的HBV基因型分布、PreS区基因突变位点的发生率,探讨HBV基因型、PreS区基因突变与CSHB发生的关系。
结果：
(1)HBV基因型研究
1)CHB组、LC组、HCC组、CSHB组的HBV基因型分布的组间差异显著,有统计学意义(P<0.05), CHB组、CSHB组以B基因型为主,LC组、HCC组以C基因型为主,进一步两两比较显示：CSHB组与LC组、CSHB组与HCC组差异显著,均有统计学意义(P<0.01)。
2)B基因型与C基因型间的临床资料比较：CHB组中各指标在两基因型间的差异不明显,均无统计学意义(P>0.05)；LC组中,C基因型中的HBV DNA和PLT水平高于B基因型(P<0.05和P=0.071); HCC组中,C基因型中的PLT水平更高、Cr水平更低,两基因型间差异明显,均有统计学意义(P<0.05)；CSHB组中,C基因型中的HBeAg阳性率更高,TBA、ALT、ALB、CHE水平偏低,差异较为显著,但均无统计学意义(P值依次为：0.071、0.064、0.054、0.055和0.093)。
3)基因B型与基因C型在CSHB患者的临床分期、临床疗效、死亡率上比较,差异均无统计学意义(P>0.05)。
4) CSHB组的HBV基因型纵向对比显示：9例临床治愈的患者中有2例发生基因型B→B/C→C型的改变。
(2) HBV PreS区基因突变研究
1)成功扩增PreS区基因片段的62份血清样本中,61份均发生了点突变,检出率在前5位的突变位点分别是：A2873(93.5%)、T3041(91.9%)、G3150(93.5%)、 A46(93.5%)、A49(64.5%)。四组患者的PreS区突变位点总数为：HCC组>CSHB组>LC组>CHB组,四组间的差异显著有统计学意义(P<0.01)。组间两两比较：LC组与CSHB组比较无统计学差异(P>0.05),其余均存在显著性差异(P＜0.01)。
2)比较CSHB组与CHB组PreS区基因突变：A2875、C2901、C2910、C2922、A2951、G2962、A2964、C2980、 G2988、G3006、G3021、C3057、T3060、G3063、 A3066、G3069、C3097、G3102、T3108、G3156、T3169、A3171、G3186、C3191、 A20、T25、A27、C40、A76、G85、G87、C93、A96、A99、T105、G110、A127、 A132、T147共39个位点的突变率有统计学差异(P<0.01或P<0.05),OR值均大于1。
3)比较CSHB组与LC组PreS区基因突变：A2946、C3009位点突变率有统计学差异(P<0.05),OR值小于1。
4)比较CSHB组与HCC组PreS区基因突变：C3009、G3156位点突变率有统计学差异(P<0.05),OR值小于1。
5) PreS1区和PreS2区起始密码子缺失和突变分析：CSHB组中：PreS1区2例(8.3%,2/24), PreS2区8例(33.3%,8/24)；CHB组中：PreSl区、PreS2区各1例(4.2%,1/24)；LC组中：PreS2区2例(20%,2/10)；HCC组中：PreS2区3例(75%,3/4)。四组患者均以PreS2区起始密码子缺失突变为主,CSHB组、HCC组的突变率都明显高于CHB组(前者P<0.05,后者P<0.01),OR值均大于1。
6) CSHB死亡组与存活组在PreS区基因突变数及点突变率上比较,均无统计学差异(P>0.05)。
7) CSHB组的PreS区基因突变纵向对比显示：3例CSHB患者在治疗前后突变位点数发生明显改变,CSHB-6:0周11个→4周70个；CSHB-9:0周10个→4周64个→24周9个；CSHB-15:0周69个→12周11个。
结论：
(1) CSHB患者的HBV基因型以B基因型为主,B基因型与C基因型的疾病严重程度相似,不同基因型对患者的临床疗效及最终结局的影响无明显区别。
(2)在治疗过程中,9例临床治愈的CSHB患者中,有2例出现HBV基因型由B→B/C→C的改变,基因型改变无加重患者病情。
(3) CSHB组的PreS区基因突变数及多个位点的突变率明显高于CHB组,PreS区变异与CSHB发生密切相关。
(4) CSHB患者的PreS区基因突变率在治疗期间可呈动态变化,在疾病进展期高,随着病情的好转,突变率下降,突变位点逐渐恢复。
Severe hepatitis is a kind of acute clinical critical disease caused by a variety of reasons with acute necrosis of massive hepatic cells and severe liver dysfunction so that liver function suddenly reduce or disappear, coagulation disorders, clinical manifestations as hepatic encephalopathy, renal damage and multiple organ failure. In China,90.29%of severe hepatitis is chronic severe hepatitis B (CSHB), with complicated clinical syndromes, variable complications, difficulty in treatment, high mortality rate, poor prognosis, continuous onsets in survivors, and the fatality was reported about60%to80%. In recent years, we have considerable advantage in researches into integrating therapy of Chinese and Western medicine, and make the death rate decline to a certain extent.
Previous studies suggested that hepatitis B virus (HBV) genotype and HBV nucleotide mutations may be related to exacerbations of hepatitis B hepatitis, but the relationship between the occurrence of CSHB and HBV mutations in patients with chronic hepatitis B is not clear. The existing research suggested that the mutations in PreS area may be involved in occurrence of CSHB, but also not conclusive.
Therefore, we carried out the following studies in this paper. In the clinical research part, clinical efficacy of integrated therapy of Chinese and Western medicine in patients with CSHB was observed. In the experimental research part, the relationship among HBV genotype, gene mutations in PreS area and the occurrence of CSHB was studied.
Part one Clinical research
Objective:
To observe the clinical efficacy of integrated therapy of Chinese and Western medicine for patients with CSHB.
Method:
Thirty-six patients with CSHB from the Eighth People's hospital during March2010to August2011were randomly divided into two groups treated with the integrated therapy of Chinese and Western medicine (treatment group,19cases) or western medicine only (control group,17cases). The main laboratory index, incidence of complications, score of traditional Chinese medical syndromes (TCM score) and clinical efficacy were monitored before and after4weeks treatment. The mortality was observed at the end of the follow-up and the death related factors of CSHB were analyzed.
Results:
(1) The total bilirubin (TBIL) and prothrombin time activity (PTA) of treatment group were significantly improved after treatment compared with that of the pre-treatment (P<0.05). The treatment group show better effect in reducing TBIL compare with the control group (P<0.05). The alanine aminotransferase (ALT) and albumin (ALB) of both groups were obviously improved after treatment compared with that of the pre-treatment (P<0.01or P<0.05), but there was no significantly difference between groups (P>0.05). The total bile acid (TBA) and cholinesterase (CHE) showed no significant change in both groups before and after treatment (P>0.05).
(2) The blood urea nitrogen (BUN) and creatinine (Cr) were in normal range before and after treatment, there was no significant difference within and between groups (P>0.05).
(3) Blood ammonia (BLA) showed no significant differences within and between groups before and after treatment.
(4) Platelets (PLT) was significantly descended in control group (P<0.01), and the difference was significantly compare with treatment group (P<0.05).
(5) After treatment, the negative conversion ratio of HBV DNA in treatment group and control group were10.5%and17.6%, there was no significance variation between groups (P>0.05). The level of HBV DNA was obviously descended, significant difference showed within groups (P<0.01) but not between groups (P>0.05).
(6) The incidences of primary peritonitis and fungal infection were significantly lower in treatment group (P<0.05, P=0.09, successively).
(7) Compare with pre-post treatment, TCM score descended obviously in treatment group (P<0.01) but not in control group (P>0.05). And significant difference showed between two groups in reducing TCM score (P<0.05).
(8) Total effective rate and significant effective rate in treatment group were73.7%
and47.4%respectively, and41.2%and11.8%in the control group, after treatment. The comprehensive curative effect and significant efficiency in treatment group were statistically significant over those of control group (P<0.05).
(9) At the end of the follow-up, the mortality was21.1%in treatment group, and was41.2%in the control group, the difference between groups showed no significance (P>0.05).
(10) Univariate analyses showed that alpha-fetoprotein (AFP), Cr and TCM score contributed statistically to the clinical outcome of patients alive or dead (P<0.01or P<0.05). The death group had higher incidences of all the complications, and in fungal infection, primary peritonitis and hepatic encephalopathy were significantly higher than survival group (P<0.01).
Conclusion:
(1) Integrating therapy of Chinese and Western medicine apply in treatment of patients with CSHB was beneficial to patients with jaundice faded faster and PLT decline slower. The treatment group had better effect on improving clinical symptoms, relieving TCM scores, decreasing complications, improving clinical efficacy and reducing mortality of patients.
(2) Good prognosis in the CSHB patients with higher level of AFP before treatment, while poor prognosis with higher incidence of complications during the treatment.
Part two Experimental research
Objective:
Explore the relationship among HBV genotypes, gene mutations in PreS area and the occurrence of CSHB. And find the possible CSHB-related mutations.
Method:
One hundred and twenty HBV infected patients from the Eighth People's hospital during March2010to August2011were recruited into the present study, including43cases of patients with chronic hepatitis B (CHB),21cases of patients with liver cirrhosis (LC),20cases of patients with hepatocellular carcinoma (HCC), and36cases of patients with CSHB (the same patients in the part one). Totally232serum samples were collected and148serum samples of CSHB patients were collected pre-post treatment and during the follow-up. Nest-PCR was employed for the amplification of the HBV genotype and PreS gene, and PCR product of PreS gene was sequenced. Then the relationship among HBV genotypes, gene mutations of PreS area and the occurrence of CSHB was analyzed.
Results:
(1) Research of HBV genotype
1) The distribution of HBV genotype in CHB group, LC group, HCC group and CSHB group was significant different (P<0.05). The major HBV genotypes in CHB group and CSHB group were genotype B, and genotype C appeared primarily in LC and HCC groups. The distribution of HBV genotypes in CSHB showed significant difference with LC group and HCC group (P<0.01).
2) In CHB group, there were no significant differences in clinical materials of genotype B and genotype C (P>0.05). In LC group, the level of HBV DNA and platelets (PLT) in genotype C were obviously higher than genotype B (P<0.01, P=0.071, successively). In HCC groups, the level of PLT and Cr displayed significantly difference between genotype B and genotype C. In CSHB group, the HBeAg rate was higher and TBA, ALT, ALB, CHE were lower in genotype C (P values in the order:0.071,0.064,0.055and0.093, compare with genotype B).
3) The clinical stages, mortality and clinical effective in CSHB group showed no significant difference between genotype B and genotype C.
4) Longitudinal analysis of genotypes in CSHB group showed that genotype in two cases of nine patients with clinical cure changed from B to B/C then last to C.
(2) Research of gene mutation in HBV PreS area
1) Spot-mutations were found in61of62amplified gene segments of PreS area. Five more mutation sites were A2873(93.5%), T3041(91.9%), G3150(93.5%), A46(93.5%) and A49(64.5%). The mutations of PreS area from most to least was HCC group, CSHB group, LC group and CHB group, the difference being significant (P<0.01). All significant difference was detected between two groups (P<0.01) except between LC group and CSHB group (.P>0.05).
2) Compared CSHB and CHB group mutation showed A2875, C2901, C2910, C2922, A2951, G2962, A2964, C2980, G2988, G3006, G3021, C3057, T3060, G3063, A3066, G3069, C3097, G3102, T3108, G3156, T3169, A3171, G3186, C3191, A20, T25, A27, C40, A76, G85, G87, C93, A96, A99, T105, G110, A127, A132, T147together39units place mutation had the significance (P<0.01or P<0.05), all OR>1.
3) Compared CSHB and LC group mutation showed A2946and C3009had the significance (P<0.05), all OR<1.
4) Compared CSHB and HCC group mutation showed C3009and G3156had the significance (P<0.05), all OR<1.
5) The initiation codon deletion and mutation of PreS1and PreS2were2cases (8.3%) and8cases (33.3%) in CSHB group,1case (4.2%) and1case (4.2%) in CHB group,0case and2cases (20%) in LC group, and0case and3cases (75%) in HCC group. Compared with CHB group, the deletion and mutation rate of PreS2was significant higher in CSHB group (P<0.05) and HCC group (P<0.01), all OR>1.
6) There did not show any significant difference in PreS gene mutation between death group and survival group (P>0.05).
7) Longitudinal analysis of PreS gene mutation in CSHB group showed that the number of mutations obviously changed in three patients before and after treatment. The numbers changed from11(0week) to70(4week), from10(0week) to64(4week) then last to9(24week), and from69(0week) to11(12week) in patient of CSHB-6, CSHB-9and CSHB-15, successively.
Conclusion:
(1) In CSHB patients, genotype B appeared primarily, clinical situation, clinical efficacy, and clinical outcome were similar between genotype B and genotype C.
(2) During the treatment, two cases of nine CSHB patients with clinical cure changed from B to B/C then to C, and this change did not worsen the situation.
(3) Gene mutation of PreS area was possibly related to the occurrence of CSHB and which was significant higher than that in CHB group.
(4) Gene mutations of PreS area changed dynamically in patients with CSHB during the treatment. It appeared more in progression stage, but along with the condition improved, its number decline.
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