苦参素联合α_1-胸腺素治疗慢性乙型肝炎的疗效

目的 观察苦参素联合α1 胸腺素治疗慢性乙型肝炎的疗效。 方法 将 72例慢性乙型肝炎患者分为两组 :联合组 38例 ,用苦参素及α1 胸腺素治疗 ;苦参素组 34例 ,单用苦参素治疗。比较两组在治疗后 3、6、9个月HBeAg及HBVDNA转阴情况。 结果 治疗 3个月后 ,两组HBeAg、HBVDNA的转阴率均为 15 .9%vs14 .7% ,差异无显著性 (χ2 =0 .0 12 ,P >0 .0 5 )。治疗 6个月和 9个月后 ,两组HBeAg、HBV DNA的转阴率差异均有显著性 (6个月后分别为 36 .8%vs 2 0 .6 % ,χ2 =4 .4 0 3,P <0 .0 5 ;39.5 %vs2 3.5 % ,χ2 =4 .2 91,P <0 .0 5。 9个月后分别为 4 7.4 %vs 2 9.4 % ,χ2 =4 .874 ,P <0 .0 5 ;5 2 .6 %vs 32 .4 % ,χ2 =7.2 13,P <0 .0 1)。 结论 苦参素联合α1 胸腺素治疗慢性乙型肝炎的疗效比单用苦参素好。

胸腺素α1-胸腺五肽在大肠杆菌中的表达及表达条件优化

目的探讨胸腺素α1-胸腺五肽(Tα1-TP5)在大肠杆菌中的表达及表达条件优化。方法将化学合成的Tα1-TP5基因与原核表达载体pGEX-4T-1融合并转化至E.coliBL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统研究培养基组成、诱导时机、诱导温度、诱导剂浓度及诱导时间等条件对融合蛋白表达量的影响。融合蛋白经GST琼脂糖珠亲和色谱纯化及重组肠激酶切割后,电喷雾质谱鉴定Tα1-TP5。结果选用TB培养基,在菌体对数生长中后期加入终浓度为0.05 mmol/L的IPTG,37℃诱导5 h,GST融合蛋白的表达量最高,占菌体总蛋白的35.8%,且主要以可溶形式表达。Tα1-TP5的分子质量与理论值相似。结论 Tα1-TP5成功在E.coli中表达,并确定了最佳表达条件。

HPLC测定胸腺素α1-胸腺五肽融合肽的含量

目的建立高效液相色谱法(HPLC)测定胸腺素α1-胸腺五肽融合肽含量的方法。方法采用Agilent ZORBAX 80A Extend-C_(18)(250 mm×4.6 mm,5μm)色谱柱,以0.05 mol/L磷酸氢二钠溶液(用磷酸调p H至7.5)-乙腈(9:1,v/v)为流动相,进样量为20μl,检测波长为208 nm,流速为0.6 ml/min;以峰面积归一化法计算含量。结果胸腺素α1-胸腺五肽融合肽在0.479~1.367 mg/ml范围内线性关系良好(r=0.9998),定量限为19.14 ng/ml,检出限为6.38 ng/ml。结论该方法专属性强,重复性和精密度均较好,结果准确,可用于测定胸腺素α1-胸腺五肽融合肽的含量。

替比夫定联合胸腺素-α1与单用替比夫定治疗HBeAg阳性慢性乙型肝炎的比较研究

目的探讨替比夫定联合胸腺素-α1治疗HBe Ag阳性慢性乙型肝炎的疗效及安全性。方法 106例HBe Ag阳性的慢性乙型肝炎患者随机分为替比夫定联合胸腺素-α1治疗组(联合用药组)52例和单用替比夫定治疗组(单用药组)54例,观察两组治疗24周、48周时ALT复常率、HBV-DNA转阴率、HBe Ag转阴率、HBe Ag血清学转换率及不良反应。结果治疗24周时,联合用药组和单用药组HBV-DNA转阴率分别为75.0%(39/52)、77.8%(42/54),差异无统计学意义(P>0.05);联合用药组HBe Ag转阴率为26.9%(14/52),HBe Ag血清学转换率为25.0%(13/52)与单用药组的18.5%(10/54)、16.7%(9/54)相比较,差异均无统计学意义(P>0.05)。治疗48周时,两组HBV-DNA转阴率比较差异无统计学意义[88.5%(46/52)、79.6%(43/54),P>0.05];联合用药组的HBe Ag转阴率为38.5%(20/52),HBe Ag血清学转换率为36.5%(19/52),与单用药组的20.4%(11/54)和18.5%(10/54)相比较,差异有统计学意义(P<0.05)。治疗过程中,两组均未发生严重不良反应。结论替比夫定联合胸腺素-α1治疗HBe Ag阳性慢性乙型肝炎疗效优于单用替比夫定,是安全有效的治疗方法。

胸腺素-α1联合干扰素-α治疗181例慢性乙型肝炎的临床观察

目的探讨胸腺素联合干扰素-α1b(IFN-α1b)治疗慢性乙型肝炎(CHB)的临床疗效。方法181例CHB患者被随机分为胸腺素联合IFN-α1b组(A组)60例、IFN-α1b组(B组)58例和非抗病毒组(C组)63例。A组给予胸腺素1.6mg皮下注射,每周2次,连续24周,干扰素-α1b5MU,肌肉注射,每日一次,连续2周后,改为隔日1次,连续22周;B组给予干扰素-α1b,方法同A组;C组只给予护肝治疗。治疗结束和随访24周时观察分析疗效。结果治疗结束时,A、B、C三组ALT复常率分别为81.7%、77.6%、74.6%,随访结束时,分别为78.3%、62.1%、50.8%,治疗结束时,HBVDNA阴转率分别为65.0%、43.1%、4.8%,随访结束时分别为61.7%、32.7%、3.2%;治疗结束时,HBeAg阴转率分别为63.3%、36.2%、3.2%,随访24周时分别为60.0%、29.3%、1.6%。结论胸腺素联合IFN-α1b治疗CHB疗效优于单用IFN-α1b。

超声引导下经皮微波消融联合胸腺素-α1治疗原发性肝癌的临床分析

目的:探析超声引导下经皮微波消融联合胸腺素-α1治疗原发性肝癌的临床效果。方法:收治70例原发性肝癌患者,参照组:实施超声引导下经皮微波消融治疗;探析组:超声引导下经皮微波消融联合胸腺素-α1治疗;比较两种治疗方案对患者肝功能的影响与治疗不良反应发生率情况。结果:治疗后,两组患者在AST、ALB与ALT指标方面互比,有差异性(P<0.05);两组在TBIL指标互比,无差异性(P>0.05)。探析组在不良反应发生率低于参照组,有差异性(P<0.05)。结论:针对原发性肝癌疾病选择胸腺素-α1联合超声引导下经皮微波消融治疗效果理想,能提高肝脏能力,不良反应少,符合临床需求。

Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris

AIM:Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Ta1 linked by different lengths of (G4S)n(n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex?75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNa2b-(G4S)n-Tα1 (n= 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex?75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNa2b monoclonal antibody and Tal polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNa2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNa2b and immunomodulatory activity of Tal in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.

超声引导下经皮微波消融治疗原发性肝癌患者疗效分析

目的探讨超声引导下经皮微波消融联合胸腺素-α1治疗原发性肝癌(PLC)患者的疗效。方法 2011年3月~2013年3月我院收治的PLC患者104例,随机分为观察组52例和对照组52例。对照组采用超声引导下经皮微波消融术治疗,观察组于术后皮下注射胸腺素-α1 4周。比较治疗前和治疗后外周血T淋巴细胞亚群、肝功能、甲胎蛋白(AFP)水平变化和两组肿瘤复发或转移及3年生存情况。结果在治疗4周时,观察组外周血CD3^+、CD4^+、CD4^+/CD8^+比值分别为71.49±6.57%、43.12±2.89%、15.89±3.24,显著高于对照组的43.21±3.74%、24.56±2.36%、5.42±2.13(P<0.05);观察组血清白蛋白(ALB)为39.84±2.56 g/L,显著高于对照组的34.12±1.87 g/L,丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、AFP水平分别为34.87±3.08 U/L、43.39±2.08 U/L、85.42±10.42μg/L,显著低于对照组的61.39±3.64 U/L、56.74±3.46 U/L、164.29±14.35μg/L;随访3年,观察组肿瘤复发率和转移率分别为30.8%和15.4%,显著低于对照组的50.0%和32.7%(P<0.05),观察组生存率为23.1%(12/52),显著高于对照组的5.8%(3/52,P<0.05)。结论超声引导下经皮微波消融后继续接受α-1胸腺素治疗PLC患者效果显著,可降低肿瘤复发或转移,延长生存时间,值得研究。

Cloning and Expression of Recombinant Human Thymosin in Yeast Pichia pastoris

The gene of human thymosin alpha 1(hT(1)was synthesised according to favorite codons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24E that is N-acetylserine were replaced by hT(1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thymosin α 1. And also,the Asn-Gly bond was designed to faciliate isolation of the target protein.The fusion gene was cloned into the expression vector, pPIC/9K. The constructs were transformed into HIS4 mutant strain GS115 by electroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.