IFN-α治疗慢性乙型肝炎中出现HBeAg血清学转换的预测因素分析

目的 观察慢性乙型病毒性肝炎(chronic hepatitis B,CHB)患者治疗后不同时期外周血谷丙转氨酶(alanine aminotransferase,ALT)、乙肝病毒脱氧核糖核酸(hepatitis B virus deoxyribonucleic acid,HBV DNA)、乙肝表面抗原(hepatitis B surface antigen,HBsAg)、乙肝e抗原(hepatitis B e antigen,HBeAg)滴度的变化,并探讨其预测HBeAg血清学转换的价值.方法 对72例选择干扰素(interferon,IFN)治疗的HBeAg阳性CHB患者随访48周,在治疗0、12、24、48周收集患者的血清,定量检测HBsAg、HBeAg、HBV DNA、ALT水平.结果 基线ALT、HBeAg水平与治疗应答相关.治疗12、24周时HBsAg＜4.33、4.07(lg IU/ml),下降率＞65％、47％,治疗12、24周时HBeAg＜2.38、0.87(lg S/CO),下降率＞71％、95％预测治疗48时出现HBeAg血清学转换的敏感性、特异性高(均有P＜0.05).结论 IFN治疗HBeAg阳性CHB患者时,动态监测HBsAg、HBeAg的水平变化能作为预测是否出现HBeAg血清学转换的良好指标.     

花生衣煎剂预防α干扰素治疗慢性丙型肝炎患者骨髓抑制临床疗效

观察花生衣煎剂对慢性丙型肝炎患者在使用IFN-α治疗过程中所致的骨髓抑制的防治作用。方法给予36例慢性丙型肝炎患者聚乙二醇干扰素联合利巴韦林治疗,同时给予花生衣煎剂口服;18例患者只接受干扰素和利巴韦林治疗,观察治疗48 w和随访24 w的疗效。结果治疗组和对照组患者在治疗48 w和随访24 w时,病毒学应答率分别为94.4%和88.8%及88.9%和83.3%（P〉0.05）;两组患者外周血白细胞和血小板计数分别为[（5.4±1.0）×10^9/L和（249.2±37.5）×10^9/L,及（2.5±1.0）×10^9/L和（81.1±25.0）×10^9/L,P〈0.01]。结论在给予IFN-α治疗慢性丙型肝炎患者过程中,加用花生衣煎剂可减轻干扰素所致的骨髓抑制。     

Treatment of pig serum-induced rat liver fibrosis with Boschniakia rossica, oxymatrine and interferon-α

AIM: To investigate the effect of Boschniakia rossica (BR), oxymatrine (OM) and interferon-alpha (IFN-α) 1b on the therapy of rat liver fibrosis and its mechanism. METHODS: By establishing a rat model of pig serum-induced liver fibrosis, liver/weight index and serum alanine transaminase (ALT) were observed to investigate the therapeutic effect of BR,OM and IFN-α. Radioimmunoassay was utilized to measure procollagen type Ⅲ (PCⅢ) and collagen type Ⅳ (CIV). RT-PCR was used to assay the expression of liver transforming growth factor- beta 1 (TGF-β1) mRNA. Immunohistochemistry of alpha-smooth muscle actin (α-SMA) and pathologic changes of liver tissues were also under investigation. RESULTS: Serum PCⅢ and CIV in BR, OM and IFN-α groups were significantly declined compared with those in model group, and their RT-PCR revealed that TGF-β1 mRNA expression was also reduced more than that in model group. Immunohistochemistry demonstrated that α-SMA also declined more than that in model group. Serum ALT in IFN-α, control and model groups was within normal level. Serum ALT in BR group had no significant difference from those of IFN-α, control and model groups. Serum ALT in OM group was significantly higher than those in BR, IFN-α, model, and control groups. CONCLUSION: BR, OM and IFN-α can prevent pig serum-induced liver rat fibrosis by inhibiting the activation of hepatic stellate cells and synthesizing collagen. OM has hepatotoxicity to rat liver fibrosis induced by pig serum.     

动态观察血清HBV-DNA水平预测干扰素治疗慢性乙型肝炎疗效的意义

目的　研究干扰素 α 2a治疗慢性乙型病毒性肝炎 (慢性乙肝 )期间 ,患者血清中HBV-DNA定量的动态变化与疗效的关系。方法　 5.8例慢性乙肝患者皮下注射干扰素 α-2a3MIU 次 ,每周 3次 ,疗程 6个月。观察患者血清中HBV DNA定量和丙氨酸转氨酶 (ALT)的动态变化。结果　完全有效组治疗 1个月后 ,患者血清中HBV DNA定量显著降低 [(3 99± 0.91)log1.0 ],明显低于部分有效组[(5.6 3± 1.31)log1 0 ]和无效组 [(6.6 9± 1.4 2 )log1 0 ],(P <0.0 5 )。疗效不同的 3组患者经过 1个月的治疗 ,血清中HBV DNA定量分别下降 (2.5 0± 0.4 4 )log1 0 、(1 6 2± 1.12 )log1 0 和 (1 0 5± 1.35 )log1 0 。通过多因素分析 ,用干扰素 α-2a治疗 1个月后患者血清中HBV DNA阴转 ,提示干扰素 α-2a的疗效好 ;治疗前ALT高水平和无家族史也与其疗效好相关。结论　患者治疗 1个月后血清中HBV DNA定量是预测干扰素 α 2a疗效的重要因素     

Establishment and characterization of a novel CD34-positive human myeloid leukemia cell line: MHH225 growing in serum-free culture

A new human multilineage myeloid leukemia cell line, MHH225, has been established in our laboratory from the bone marrow of a 60-year-old patient suffering from acute megakaryoblastic leukemia (M7); it provides a unique model for studying the effect of biologic and chemical agents on the lineage specificity of a multipotent myeloid leukemia clone containing a mixed population of megakaryoblast, erythroblast, and myeloblast cells in a serum-free culture. Morphologically, all 225 cells are large blast cells with basophilic cytoplasm containing no granules, large round nucleus containing 2–3 prominent nucleoli, and fine chromatin structure and a large nuclear/cytoplasm ratio. The MHH225 cells are CD34⁺HLA-DR⁺CD33⁺CD13⁺ with 57.6%, 28.3%, and 7.8% of them being CD41⁺, glycophorin A⁺, and CD15⁺, respectively, and all lymphoid-specific antigens are negative. The karyotype analysis of MHH225 cells revealed a deletion of the short arm of chromosome 7: del(7)(p13)-, a whole-arm translocation between the long arms of chromosomes 9 and 21: t(9;21)(q10;q10), and a chromosome 11 with an elongated long arm due to duplication of chromosome 11 material as well as to translocation of part of chromosome 9 onto 11q+. Also, chromosome 21 was deleted in some metaphases or showed a ring formation in other metaphases. Utrastructurally, MHH25 cells display a strong platelet peroxidase activity in the nuclear envelope and the endoplasmic reticulum. The MHH25 cells have been grown exponentially without growth factors or conditioned media or serum only in RPMI1640 culture medium. None of the myelopoietic growth factors, i.e., interleukin-3, GM-CSF, G-CSF, erythropoietin, or interleukin-6, has any effect on the proliferation and differentiation of MHH25 cells. The two, hematopoietic inhibitory cytokines, interferon-alpha and tumor necrosis factor-alpha, have only minimal growth inhibitory effect. Stem cell factor showed only weak growth-stimulatory effect on MHH225 cells but significantly inhibited chemotherapy-induced apoptosis in these cells. The new cell line MHH225 should constitute a useful model for studying stem cell antigen (CD34)-positive human multilineage myeloid leukemia cells carrying a deletion in the short arm of chromosome 7 and an aberration in chromosome 11 and provide a unique tool for investigating human hematopoietic stem cell biology and its cytokine regulation in serum-free cultures. To our knowledge, the MHH225 cell line is the first human CD34-positive leukemia cell line growing in serum-free cultures to be established.     

慢性乙型肝炎干扰素治疗后长期随访复发与再治疗后复发研究

目的观察和分析慢性乙型病毒性肝炎（CHB）经重组α干扰素（rIFN-α）治疗取得联合应答后,在长期随访过程中的复发情况,及再次rIFN-α治疗后的复发情况。方法523例经肝穿刺活检证实的CHB患者,给予rIFN-α1b治疗,每次500万u,每周3次,疗程6～37个月（中位数10个月）,治疗中每1～3个月检测肝功能、HBVDNA、乙肝炎e抗原（HBeAg）。治疗后随访至少12个月,随访时每3～6个月检查肝功能、HBVDNA、HBeAg,随访中的复发病例,予以第2次rIFN-α治疗。结果523例患者HBeAg（＋）403例,HBeAg（-）120例。初次rIFN—α治疗结束后,HBeAg（＋）组近期应答225例（55．8％）,HBeAg（-）组77例（64．2％）,差异无统计学意义（Χ^2=2．633,P=0．105）。302例近期应答者,经随访（39±22）个月,复发119例（39．4％）,其中HBeAg（＋）组76／225例（33．8％）、HBeAg（-）组43／77例（55．8％）,差异有统计学意义（Χ^2=19．335,P=0.000）。按随访每12个月为一时间段,分1～12个月、13～24个月、25～36个月、37～48个月、49～60个月和≥61个月6个时间段,各时间段复发的发生率差异有统计学意义（Χ^2=73．518,df=5,P=0．000）,累计复发率差异亦有统计学意义（Χ^2=32．167,df=5,P=0．000）。各时间段复发发生的HBeAg阳性比差异无统计学意义,Χ^2=2．518,df=4,P=0．641,累计复发的HBeAg阳性比差异亦无统计学意义,）（Χ^2=0．370,df=5,P=0．996。57例复发者[HBeAg（＋）组25例,（-）组32例]接受第2次rIFN-α治疗,治疗结束时全部取得联合应答,但HBeAg（＋）组13例,（-）组7例第2次复发,差异有统计学意义（Χ^2=5．592,P=0．018）。结论CHB患者经初次rIFN-α治疗后,HBeAg阴性组与HBeAg阳性组的近期应答率相同,而HBeAg阴性组的复发率高于HBeAg阳性组。复发者第2次rIFN-α治疗结束时可全部取得联合应答,而第2次复发HBeAg阴性组低于HBeAg阳性组。     

Association of IL28B Polymorphisms With the Response to Peginterferon Plus Ribavirin Combined Therapy in Polish Patients Infected With HCV Genotype 1 and 4

Background

Three single nucleotide polymorphisms (SNPs) near interleukin-28B (IL-28B) gene were shown to be highly associated with treatment response (SVR) in patients with chronic hepatitis C virus (HCV) infection. There is limited data about the role of single and combined IL-28B polymorphisms in HCV-infected Polish population.

Objectives

This study''s aim was to determine predictability of three IL-28B gene polymorphisms and other known prognostic factors on the treatment response in HCV genotype 1 and 4 infected Polish patients. The effect of IL-28B polymorphisms on therapy was also compared with other known prognostic factors.

Patients and Methods

We genotyped IL-28B polymorphisms (rs12979860, rs12980275 and rs8099917) by polymerase chain reaction-based restriction fragment length polymorphism assay in a group of 293 patients from which a selected cohort of 174 treatment-naiev patients underwent treatment.

Results

We showed that rs12979860 CC [odds ratio (OR) = 4.6, P < 0.001], rs12980275 AA (OR = 2.9, P = 0.002) and rs8099917 TT (OR = 2.2, P = 0.016) genotypes were associated with successful treatment compared to the rs12979860 CT-TT, rs12980275 AG-GG and rs8099917 TG-GG, respectively. Patients bearing of IL-28B profile including the three favourable genotypes do not have much chance of a recovery (OR = 3.4, P = 0.002). Except for IL-28B polymorphisms, there was no association of SVR with any other pretreatment clinical data in analyzed group. The correlation of SNPs with other host and viral factors revealed association of favorable genotypes of IL-28B markers with high levels of alanine aminotransferase and baseline HCV viral load.

Conclusions

IL-28B polymorphisms were the strongest pretreatment predictors of response to pegylated interferon and ribavirin in Polish patients chronically infected with HCV genotype 1 and 4. This study confirm the strongest impact of IL-28B rs12979860 on SVR, nevertheless rs12980275 AA seems to be more important than rs8099917 TT in predicting positive treatment response.     

联合干扰素-α和吉非替尼对结肠癌细胞的生物学作用研究

目的 探讨干扰素(IFN)-α、吉非替尼对人结肠癌细胞系HCT116的增殖、凋亡作用.方法 采用结肠癌细胞HCT116为研究对象,观察不同浓度IFN-α(50 U/ml和100 U/ml)、吉非替尼(0.5 μmol/L,2.5 μmol/L和5.0 μmol/L)单独和联合应用(IFN-α 50 U/ml+吉非替尼0.5 μmol/L)和不同作用时间点(作用24、48和72 h)对细胞的生物学作用.四甲基偶氮唑盐(MTT)法检测药物对细胞的增殖抑制作用,光学显微镜观察细胞形态变化,流式细胞学检测细胞凋亡情况.采用SPSS 13.0软件进行统计学分析,两组间比较采用t检验,多组数据比较采用单因素方差分析.结果 IFN-α、吉非替尼单独和联合应用均能明显抑制HCT116的增殖(P值均 <0.05),且抑制程度与药物作用时间和药物浓度之间存在依赖效应.药物作用下可观察到细胞呈现凋亡形态学改变,流式细胞学结果显示HCT116细胞凋亡率明显增加,IFN-α(50 U/ml)、吉非替尼(0.5 μmol/L)单独和联合应用72 h后细胞凋亡率分别为15.6%±0.6%、13.6%±0.4%和31.2%±0.3%明显高于对照组的6.8%±0.3%,差异均有统计学意义(P值均<0.01).结论 IFN-α、吉非替尼均能抑制HCT116细胞生长并诱导细胞凋亡,两者联合应用具有协同增效作用.

Abstract：

Objective To investigate the effects of interferon-α (IFN-α) and gefitinib on the proliferation and apoptosis of human colon cancer cell line HCT116. Methods Colon cancer cell line HCT116 was selected as research objective. The biological effects of IFN-α and gefitinib alone or combined on the cells were observed at different time point (after worked for 24, 48 and 72 hours). The proliferation inhibition of the medicine on the HCT116 cells was measured by methyl thiazolyl tetrazolium (MTT) assay. Morphologic changes were observed under optical microscope. Apoptosis was measured by flow cytometry (FCM). The results were analyzed with SPSS 13.0 software, two groups compare was tested by t test, and single factor variance analysis was for multiple group data compare. Results IFN-α and gefitinib alone or combined could significantly inhibit the proliferation of HCT116 cells (P<0.05), and there was a time and dose-dependent manner between the degree of inhibition and the working time and concentration of the medicine. With the work of the medicine, apoptosis morphologic changes were observed in the cells. And FCM result indicated that the apoptosis rate significantly increased. After treated with IFN-α and gefitinib alone or combined for 72h, the cell apoptosis rate were 15.6%±0.6%, 13.6%±0.4% and 31.2%±0.3% respectively, which was obviously higher than control group (6.8%±0.3%, P<0.05). Conclusion Both IFN-α and gefitinib were able to inhibit the proliferation and induce apoptosis of HCT116 cells moreover, and a synergistic effect was observed while combine used there two medicines.     

乙型肝炎病毒基因型、基础核心启动子和C区变异与干扰素疗效的观察

目的 探讨HBV基因型、基础核心启动子(BCP)和C区变异与IFNα抗病毒疗效的相关性.方法 选择IFNα-1b治疗6个月的HBeAg阳性慢性乙型肝炎(CHB)患者,随访6个月.应用限制性片段长度多态性(RFLP)法检测HBV基因型,PCR测定BCP、前C/C区核苷酸序列.计量资料采用t检验、方差分析,计数资料采用卡方检验、Fisher确切概率法,并进行多因素条件Logistic回归分析.结果 共39例CHB患者完成观察,治疗结束时,应答16例,占41.0%;随访结束时,应答者中持续应答12例,占30.8%,复发4例,占10.3%.其中B基因型29例,占74.4%,C基因型10例,占25.6%.基因型型别差异与IFNα-1b疗效无关.8例患者BCP区T1762/A1764双变异,占20.5%,与IFNα-1b疗效无相关.8例患者检出A1896变异,占20.5%,随访结束时获得疗效的3例A1896变异株感染者均复发.C区非淋巴细胞表位测序发现,15例患者L60V变异,占38.5%,14例为I97L变异,占35.9%.与60V比较,表现为60L的患者在随访结束时的HBeAg血清学转换率和HBV DNA阴转率明显低(Fisher确切概率法,P=0.0126、0.0069).与97L比较,表现为97I的患者在治疗结束时和随访结束时的HBV DNA阴转率明显低(Fisher确切概率法,P=0.0484、0.0024).Logistic回归分析显示,基因型、C区变异与IFNα-1b疗效无相关.结论 HBV基因型、BCP双变异与IFNα的疗效无关,C区非淋巴细胞表位L60V及I97L变异可能有利于IFNα的治疗.

Abstract：

Objective To investigate the association between hepatitis B virus (HBV)genotype, the mutations in HBV basic core gene promoter(BCP), pre C/C gene region and treatment response to interferon (IFN)α-1b. Methods Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients were treated with IFNα-Ib for 6 months and were followed up for 6 months after the end of treatment. Restriction Fragment Length Polymorphism (RFLP) was used for determining HBV genotype. HBV DNA was amplified by polymerase chain reaction (PCR) and analyzed for BCP and pre C/C gene region by sequencing. Measurement data were compared using t test and analysis of variance. Enumeration data were compared using chi-square test, Fisher exact probability test.Logistic regression analysis was utilized for multi-factor analysis. Results There were 39 patients who completed the treatment and follow up in this study. At the end of treatment, 16(41.0%) patients showed response to the IFNα-lb treatment. At the end of follow-up, four out of 16 patients who achieved on treatment response relapsed. Among 3a patients, 29 (74.4 %) were infected with genotype B and 10 (25. 6%) with genotype C. The treatment response rates were not significant different between the groups with different genotypes. The double mutation pattern (T1762/A1764) was found in eight (20. 5%) patients. The response rates to IFNα-lb treatment were not significant different between the group with and without double mutation pattern. A1896 mutation was detected in eight patients at baseline. Three of them became HBeAg negative at the end of treatment and returned to HBeAg positive during follow-up. The non-lyphocyte epitope mutations, L60V and I97L, were found in 15 patients (38. 5%) and 14 patients (35.9%), respectively. At the end of follow-up, the patients with 60V had a significantly lower HBeAg seroconversion rate and HBV DNA undetectable rate compared to the patients with 60L (Fisher exact probability test; P = 0.0126 and 0.0069,respectively). The HBV DNA undetectable rates in the patients with 97I were significantly lower than those in patients with 97L both at the end of treatment and the end of follow-up (Fisher exact probability test; P= 0.0484 and 0. 0024, respectively). Logistic regression analysis results showed that there was no association between the above viral mutations and the treatment response to IFNαlb. Conclusions There is no association between HBV genotype, BCP double mutation pattern and IFN-α treatment response. The non-lyphocyte epitope mutations, L60V and I97L, may have impact on IFN-α treatment response.     

两种具长效性重组人血清白蛋白／干扰素α融合蛋白表达工程菌的构建及融合蛋白的理化特性研究

目的对两种长效干扰素α融合蛋白原料药的理化特性多项指标开展分析和进行比较。方法利用基因工程技术分别构建了可高效表达重组人血清白蛋白／干扰素α2a融合蛋白（rHSA／IFNα2a）或重组人血清白蛋白,干扰素α2b融合蛋白（rHSA／IFNα2b）的毕赤酵母工程菌。融合蛋白直接分泌到组分简单的无机盐培养基中,并经特别建立的高效分离纯化工艺进行纯化,随后对两个融合蛋白进行详尽的理化特性分析。结果获得的融合蛋白纯度在98％以上;N端前15个氨基酸序列与理论序列相同;质谱分子量分别为85640．8D和85781．5D;圆二色光谱分析比对蛋白空间构象未变;等电点DI约在5．1左右;为非糖基化蛋白;紫外光谱呈典型蛋白质光谱;自由巯基含量测定值为1．4;肽图批次问一致;肽质量指纹图谱可匹配分别达83％和82％;对制备的2个融合蛋白原料药中的细菌内毒素、宿主蛋白质、外源性DNA、甲醇和甘油的残留量检测符合标准要求;融合蛋白的免疫学鉴别为阳性;体外细胞生物比活性约为2．5×10^5IU／mg,并且2个融合蛋白生物比活性测定值无不同,由此,获得了符合临床用药标准的原料药。结论研究所获得的结果可以作为指导《注射用重组人血清白蛋白／干扰素α2a融合蛋白》和《注射用重组人血清白蛋白／干扰素α2b融合蛋白》2个新药的原料药生产质量标准的建立和检定方法的确定。