Acute Toxicity of Recombinant Porcine Interferon-alpha

To observe the acute toxicity of recombinant porcine interferen-alpha （IFN-alpha） in mice and thus provide a basis for the clinical safety. [Method] According to the principles of acute toxicity, all the mice were divided into two major groups （intraperitoneally injected group and intramuscularly injected group） respectively at high dose, moderate dose and low dose. And the normal control group was also set up. Within 14 d after administration, the behavior of mouse and the degree of toxicity were continuously observed. The hematological indexes and biochemical indexes of blood were detected to obtain the preliminary toxicity data of the recombinant porcine IFN-alpha. And at the end of the experiment, mice were sacrificed for autopsy. [ Result] There was not significant difference in external performance, behavioral characteristics, body temperature, weight, pathological anatomy of visceral organs, hematological indexes and biochemical indexes between the experimental groups and the control group. [ Conclusion] The highest dose of porcine interferon （5.0 x 10s IU per mouse） in this experiment or the dose lower than this dosage should not have significant toxic effects on mice, and the recombinant porcine IFN-alpha is safe in clinical application.

Relationship between HBV genotypes and anti-viral therapeutic efficacy of interferon-alpha

BACKGROUND: Much evidence demonstrates that the genotypes of hepatitis B virus (HBV) present differences in pathogenicity and outcomes owing to differences in genetic structure. This study aimed to investigate the influences of HBV genotypes on the anti-viral therapeutic efficacy of interferon-alpha (IFN-alpha) in chronic hepatitis B patients, and to determine the relationship between HBV genotypes and levels of viral replication or gene variations. METHODS: The chronic hepatitis B patients who were treated with IFN-alpha were selected randomly. Anti-viral therapeutic efficacy was monitored in these patients. The HBV genotypes were detected by PCR microplate hybridization ELISA. The levels of serum HBV-DNA were determined by fluorescence quantitative PCR. HBV gene variation at pre-C and basic core promoter (BCP) regions were assayed by gene chip technology. RESULTS: Genotypes B and C were predominant in 94 chronic hepatitis B patients. A, E and F genotypes were not found in these patients. The HBV-DNA levels of genotype C and mixed genotypes were significantly higher than those of genotype B. The response to IFN-alpha in patients with genotype B was markedly better than in those with genotypes C and D, and the complete response to IFN-alpha was only observed in genotype B. The response to IFN-alpha in patients with mixed genotypes was the least sensitive. The negative transition of HBeAg was correlated with variations in the HBV pre-C and BCP regions in patients with partial or no response to IFN-alpha. The variation rates of HBV pre-C and BCP regions were clearly higher in genotype C than in genotype B. CONCLUSIONS: The results suggest that HBV genotype is correlated with the serum levels of HBV-DNA, HBV gene variations and therapeutic efficacy of IFN-alpha. The regular detection of HBV genotypes in the clinic will be of benefit for disease prognosis and planning of anti-viral therapeutic strategies.

Simultaneous development of diabetic ketoacidosis and Hashitoxicosis in a patient treated with pegylated interferon-alpha for chronic hepatitis C

Classical interferon-alpha has been shown to be correlated with the development of a variety of autoimmune disorders. A 38 year-old female patient developed simultaneously diabetic ketoacidosis and hyperthyroidism 5 mo following initiation of treatment with pegylated interferon-α and ribavirin for chronic hepatitis C. High titers of glutamic acid decarboxylase, antinuclear and thyroid (thyroid peroxidase and thyroglobulin) antibodies were detected. Antiviral treatment was withdrawn and the patient was treated with insulin for insulin-dependent diabetes mellitus and propranolol for hyperthyroidism. Twelve months after cessation of pegylated interferon-α therapy the patient was euthyroid without any medication but remained insulin-dependent.

T29C genotype polymorphism of estrogen receptor alpha is associated with initial response to interferon-alpha therapy in chronic hepatitis B patients

BACKGROUND: Virological clearance, delayed progression to cirrhosis or liver cancer, and increased survival are the long-term goals of antiviral therapy in chronic hepatitis B patients. Identification of host factors correlated with therapeutic response may contribute greatly to individual treatment. This study aimed at investigating whether T29C genotype polymorphism of estrogen receptor alpha (ESR1) is associated with the initial response to interferon-alpha (IFN-alpha) therapy in chronic hepatitis B patients. METHODS: The initial responses of 100 patients to IFN-alpha therapy were evaluated and compared by classifying them into three groups according to T29C genotype polymorphism of ESR1: T/T, TIC, and C/C genotype groups. Polymerase chain reaction-restriction fragment length polymorphism was used to analyze the genotype polymorphism in T29C. RESULTS: The frequency of initially combined response was markedly higher in both the T/T and TIC groups than in the C/C group (Z=10.326, P=0.006 and Z=26.247, P=0.000, respectively). In addition, the initial virological response was higher in the T/T and T/C groups than the C/C group (chi(2)=5.674, P=0.017 and chi(2)=4.980, P=0.026, respectively). In 78 initially HBeAg-positive patients, however, the frequency of initial e-antigen disappearance or seroconversion among the T/T, T/C, and C/C genotype groups was 34.15%, 27.78% and 15.79%, respectively, which were not significantly different. CONCLUSION. The T29C genotype polymorphism of ESR1 is associated with the initial response to IFN-alpha in patients with chronic hepatitis B, and might be a significant marker for predicting the initial response to IFN-alpha, at least in this study population. (Hepatobiliary Pancreat Dis Int 2010; 9: 275-279)

Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

AIM: To investigate the effects of interferon-alpha (IFN-α) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein. METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-α. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/ pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx. RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-α. CONCLUSION: IFN-α can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-α therapy of HCC.

Abrupt onset of type 1 diabetes mellitus during recombinant interferon-alpha 2b therapy in a patient with chronic hepatitis B

We describe a case of a 33-year-old female patient with chronic hepatitis B who developed type 1 diabetes mellitus (DM) after a 13-mo period of treatment with recombinant human interferon-alpha (IFN-α) 2b. The patient presented with polydipsia, polyuria, hypergly-cemia, diabetic ketoacidosis, combined with C-peptide secretion defi ciency and positive islet cell autoantibody (ICAb). IFN-α 2b treatment was terminated and in-stead insulin treatment was initiated. Five months after cessation of the recombinant human IFN-α 2b therapy, the patient remained insulin-dependent. Her serum HBV DNA became negative and serum transaminase returned to the normal level after a 10-mo period of IFN therapy. Type 1 DM induced by IFN-α is relatively rare in patients with chronic hepatitis B. We should pay more attention to patients on IFN-α therapy to avoid destruction of pancreatic beta cells. This is the first case report from China.

Gastric autoimmune disorders in patients with chronic hepatitis C before,during and after interferon-alpha therapy

AIM:To explore the prevalence of autoimmune gastritis in chronic hepatitis C virus (HCV) patients and the influence of α-interferon (IFN) treatment on autoimmune gastritis. METHODS:We performed a prospective study on 189 patients with positive anti-HCV and viral RNA enrolled in a 12-month IFN protocol.We evaluated:a) the baseline prevalence of autoimmune gastritis,b) the impact of IFN treatment on development of biochemical signs of autoimmune gastritis (at 3,6 and 12 months),c) the evolution after IFN withdrawal (12 months) in terms of anti-gastric-parietal-cell antibodies (APCA),gastrin,anti-thyroid,and anti-non-organ- specific antibodies. RESULTS:APCA positivity and 3-fold gastrin levels were detected in 3 (1.6%) and 9 (5%) patients,respectively,at baseline,in 25 (13%) and 31 (16%) patients at the end of treatment (both P<0.001,vs baseline),and in 7 (4%) and 14 (7%) patients 12 months after withdrawal (P=0.002 and P=0.01 respectively,vs baseline;P=not significant vs end of treatment).The development of autoimmune gastritis was strictly associated with the presence of autoimmune thyroiditis (P=0.0001),no relationship was found with other markers of autoimmunity. CONCLUSION:In HCV patients,IFN frequently precipitates latent autoimmune gastritis,particularly in females.Following our 12-month protocol,the phenomenon generally regressed.Since APCA positivity and high gastrin levels are associated with the presence of antithyroid antibodies, development of autoimmune thyroiditis during IFN treatment may provide a surrogate preliminary indicator of possible autoimmune gastritis to limit the need for invasive examinations.

Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis

BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully, rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.

Synergistic effects of interferon-alpha in combination with chemoradiation on human pancreatic adenocarcinoma

AIM: To determine whether IFN-α is the agent that turns a slightly effective treatment (radiochemotherapy) into a potent therapy, we tested IFN-α for its synergistic properties.METHODS: Eight pancreatic carcinoma cell lines were treated with the single agents and combinations of these.The role of IFN-α regarding a) direct inhibitory effects; b)radio and chemosensitizing effects; c) anti-angiogenic properties and d) enhancement of immunogenicity was investigated.RESULTS: Our results show that IFN-α has direct inhibitory properties and some synergistic influence as determined by AnnexinV/PI stain and cell count. IFN-α is also able to prevent the increase in proliferation rate and VEGF secretion of CDDP resistant cells. Having taken the results from immunogenicity experiments together, we found cells that can be influenced by IFN-α but were less susceptible against T cells. Furthermore, high expression of MHC molecules, CD118, EGF-R and Fas was predictive for a good response.CONCLUSION: In conclusion, IFN-α has direct cytotoxic effects, acts as a radiosensitizer and circumvents tumor cell-regrowth after CDDP treatment. These mechanisms may be responsible for the good clinical outcome of CapRI.

Purification of Recombinant Porcine Interferon-Alpha

[ Objective] To study the purification of recombinant porcine interferon-alpha （rPolFN-alpha） and lay a foundation for researches on the structure of the rPolFN-alpha and the preparation of standard proteins. [ Method] The rPolFN-alpha were induced and extracted from the recombi- nant E. coil BL21, and they were purified by two strategies. The first strategy was that the rPolFN-alpha were purified by GST （ glutathione S transferase） affinity chromatography, DEAE （diethylaminoethyl） anion exchange chromatography and gel filtration in turn defined as three-step chromatography method; the second strategy was that the rPolFN-alpha were purified by GST affinity chromatography and gel filtration in tum defined as two-step chromatography method. Then the purified products were detected by the SDS-PAGE （sodium dodecyl sulfate polyacrylamide gel electrophoresis） and were identified by western-blotting. [Result] The purity quotient of pudfied products of the two-step chromatography method was 96.0% and that of the three-step chromatography method was 98.8%. The purified products were detected by the SDS-PAGE and the western- blotting, respectively. The results showed that the target band was 45.0 kDa and the specific band was found. [ Conclusion] The purity quotient of proteins of the two-step chromatography method is close to that of the three-step chromatography method, thus the two-step chromatography meth- od is more convenient and more suitable for pilot production than the three-step chromatography method.

Effect of Interferon-alpha in systemic lupus erthematosus (SLE) serum on the differentiation and maturation of dendritic cells derived from CD34^+ hematopoietic precursor cells

Objective： To study the effect of interferon-alpha IFN-α in the serum of SLE patients on the differentiation and maturation of dendritic cells （DCs） derived from CD34^＋ hematopoietic precursor cells （HPCs）. Methods： Serum samples from SLE patients and normal controls were collected and the concentration of IFN-α detected by ELISA. CD34^＋HPCs were purified from cord blood by a magnetic cell sorting system （MACS）, and cultured to differentiate to DCs. Normal serum, normal serum with exogenous IFN-α, SLE serum with raised levels of IFN-α, or SLE serum with anfi-IFN-α neutralizing antibody was added to the culture medium. The phenotype of DCs was analyzed by flow cytometry （FCM） and the capacity of DCs to stimulate allogenic T lymphocyte proliferation was evaluated in a mixed lymphocyte reaction by the Cell Counting Kit-8. Cytokine production was assessed by ELISA. Results： Serum levels of IFN-α were significantly higher in SLE patients than in normal controls and this correlated positively with disease activity. Cultured in SLE serum with raised levels of IFN-α, CD34^＋ HPCs could differentiate into DCs that expressed higher levels of HLA-DR, CD80 and CD86, and showed an enhanced allogenic T-cell stimulatory capacity, while producing lower levels of IL-12 and higher amounts of IL-10 compared with those DCs cultured in normal serum. Conclusion： Increased levels of IFN-α in SLE serum promotes the differentiation and maturation of DCs derived from CD34^＋ HPCs and could contribute to the pathogenesis of SLE.

Clinical correlations between chronic hepatitis C infection and decreasing bone mass density after treatment with interferon-alpha

Objective: To compare the bone mass density in chronic hepatitis patients before and after interferon-a treatment.Methods: A total of 70 patients with chronic hepatitis C were treated with interferon-a and were evaluated. The treatment dosage was three million IU three times a week for one year. All the patients underwent bone mass density detection at lumbar spine and femoral neck before and after the interferon-a treatment. All the necessary information such as age,sex, and laboratory test, history of occurrence of fractures, lifestyle, and menopause status was collected by interviewers face-to-face from participants at the research visit. Smoking was categorized by whether participants were nonsmokers or smokers. Menopause was designated if there had been complete cessation of menses for more than 12 months. All statistical analyses were performed by SPSS version 14(SPSS, Inc., Chicago, IL, USA).Results: Among 70 patients, 52% were male, 48% were female and the mean age was(57.0 ± 9.6) years(range: 24–79). Twenty-nine percent of the patients had a history of smoking. The mean body mass index was(24.4 ± 3.6) kg/m^2(range: 18.4–35.3). Of the70 cases, 21 had high fibrosis-4. The prevalence of overall fracture history was 2.9%(two patients).Conclusions: Chronic hepatitis C virus infection did increase the risk of development of metabolic bone disease in this cohort. Indeed, greater reduction of bone mass density occurs in advanced liver fibrosis. The bone loss in earlier stages of chronic hepatitis C infection is likely to result from increased bone reduction rather than decreased bone formation. Overall, these observations suggest an important role for chronic hepatitis C virus infection in increased bone turnover in osteodystrophy pathogenesis.

重组猪干扰素α发酵纯化与抗病毒活性测定

本研究根据大肠杆菌的密码子偏好性,对UniProt数据库中的猪干扰素α序列(编号:P49879)进行优化,合成了相应的基因片段,并构建了pET-28a-INF-α原核表达载体。将重组载体转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达,经过20 L发酵罐培养24 h,在OD 600为108时收获细胞。表达的猪干扰素α主要以包涵体的形式存在,经过变性、复性、镍亲和纯化与酶切等步骤,得到了无标签的猪干扰素α蛋白。SDS-PAGE电泳显示,该蛋白的分子量约为19 kDa,纯度高达95%。细胞病变抑制法测定表明,该蛋白的抗病毒活性为1.28×10^(7 )IU/mg。本研究成功获得了高纯度、高活性的猪干扰素α蛋白,为进一步推进蛋白药物在临床上的应用奠定了基础,为进一步重组蛋白工业化生产提供了理论依据。

聚乙二醇干扰素α-2b联合替诺福韦对乙型肝炎患者肝功能及安全性的影响

目的:探讨聚乙二醇干扰素α-2b联合替诺福韦对乙型肝炎患者肝功能及安全性的影响。方法:选取2018年10月—2020年8月南阳医学高等专科学校第一附属医院收治的96例乙型肝炎患者作为研究对象,采用乱数表法分为A组和B组,每组各48例。A组给予替诺福韦酯片治疗,B组在A组基础上加用聚乙二醇干扰素α-2b治疗。比较两组患者治疗前、后总胆红素(TBIL)、白蛋白(ALB)、天门冬氨酸氨基转移酶(AST)、谷丙转氨酶(ALT)、干扰素-γ(IFN-γ)、白介素-10(IL-10)、白介素-4(IL-4)和白介素-2(IL-2)水平。比较两组患者乙型肝炎病毒(HBV)-DNA转阴率、乙型肝炎e抗原(HBeAg)转阴率、转换率和ALT复常率。统计两组患者治疗有效率和不良反应发生率。结果:治疗后,B组总胆红素(TBIL)、天门冬氨酸氨基转移酶(AST)和谷丙转氨酶(ALT)水平均低于A组,两组患者白蛋白(ALB)均升高且B组升高幅度大于A组,差异有统计学意义(t=15.629、27.253、30.580、11.761,P<0.05);治疗后,两组患者酶联免疫吸附测定干扰素-γ(IFN-γ)和白介素-2(IL-2)均升高,且B组升高幅度大于A组,白介素-10(IL-10)和白介素-4(IL-4)均降低,且B组降低幅度大于A组,差异有统计学意义(t=20.777、7.614、24.624、1.609,P<0.05);B组HBV-DNA转阴率、ALT复常率、乙型肝炎e抗原(HBeAg)转阴率、HBeAg转换率均高于A组,差异有统计学意义(χ^(2)=4.381、4.800、4.019、4.909,P<0.05);B组治疗总有效率高于A组,差异有统计学意义(χ^(2)=4.360,P<0.05);两组患者不良反应发生率比较,差异无统计学意义(χ^(2)=0.079,P>0.05)。结论:聚乙二醇干扰素α-2b联合替诺福韦可有效改善患者肝功能,调节免疫功能且安全性较高。

Mindin蛋白在经聚乙二醇干扰素α-2b治疗的慢性乙型肝炎中的动态变化及意义

目的 分析聚乙二醇干扰素α-2b(PEG-IFNα-2b)治疗慢性乙型肝炎(CHB)过程中Mindin蛋白的变化以及作用。方法 选取2018年1月—2019年12月于西安交通大学第二附属医院行PEG-IFNα-2b治疗的CHB患者29例,按照临床结局分为治愈组(n=17)与未治愈组(n=12)。分别采集治愈组和未治愈组基线、12周和24周的外周血样本,测量血常规、肝功能、乙型肝炎标志物定量和Mindin蛋白含量。分析比较各时间点HBsAg、ALT、AST及Minidn蛋白水平的组间差异。符合正态分布的计量资料两组间比较采用成组t检验;非正态分布的计量资料两组间比较采用Mann-Whitney U检验。采用Spearman相关性分析法对Mindin蛋白与HBsAg、ALT、AST的相关性进行分析。多元线性回归分析探讨HBsAg、ALT水平对Mindin蛋白的影响。结果 基线资料分析发现,未治愈组和治愈组HBsAg、抗-HBe、Alb和白/球比值(A/G)水平两组间比较差异均有统计学意义(P值均<0.05)。治愈组Mindin蛋白水平呈现持续上升的趋势,24周时Mindin蛋白水平显著高于基线(P<0.05)。24周时治愈组Mindin蛋白水平显著高于未治愈组(P=0.019)。治愈组HBsAg水平均显著低于未治愈组,且组内各时间点与基线比较差异均具有统计学意义(P值均<0.05)。此外,治愈组ALT和AST的变化均呈现先升高后降低的趋势,12周的表达水平均显著高于基线(P值均<0.05)。未治愈组24周ALT和AST水平均显著高治愈组(P值均<0.05)。未治愈组12周时Mindin蛋白水平与ALT呈现出较强的直线相关性(r=0.760 8,P<0.05),进一步的多元线性回归分析同样证明两者间存在线性关系(偏回归系数为1.571,P=0.019)。结论 PEG-IFNα-2b抗病毒治疗24周时的Mindin蛋白水平在治愈组和未治愈组间存在明显差异。提示通过检测Mindin蛋白的动态变化可以更好地预测慢性乙型肝炎的治疗结局,为临床提供参考。

中国旱獭Ⅰ型干扰素受体β亚基克隆、表达及功能初步鉴定

目的克隆中国旱獭Ⅰ型干扰素受体β亚基(mhIFNAR2)的基因,并进行抗体制备及功能鉴定。方法应用RT-PCR技术,从中国旱獭脾组织中扩增得到序列,克隆至原核表达载体p RSET-B,表达重组蛋白,电泳和Western Blot法鉴定;重组蛋白常规免疫BALB/c小鼠制备其胞外段多克隆抗体,免疫组化、免疫荧光和Western Blot法鉴定;再通过si RNA阻断的方法检测其功能。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果从mhIFNAR2扩增出149~1300 bp片段,其同源性在分析的种属中以土拨鼠最高,可达98.05%。成功地构建了表达胞外段mhIFNAR2_((50-181aa))蛋白的原核表达质粒,命名为pRSET-B.mhIFNAR2;其表达重组蛋白分子量27 kD,纯化后纯度约为95%,浓度约为160μg/mL。用纯化的重组蛋白常规免疫BALB/c小鼠后,获得1∶1000的特异性多克隆抗体,用免疫组化及免疫荧光可见细胞膜、细胞质有表达。合成的三条siRNA,其中有一条起始于277位点的siRNA(siRNA277)与空白对照及阴性对照相比,可以沉默目的基因的表达,并能减弱干扰素的信号通路(P值均<0.05)。结论获得mhIFNAR2的部分序列,成功地制备出抗mhIFNAR2胞外段多克隆抗体,该抗体有较高的效价和特异性,并能用于免疫组化、免疫荧光及Western Blot的检测。用siRNA277可以抑制目的基因的表达,并能阻断干扰素的信号通路。

MicroRNA-4661 inhibits antiviral innate immune response by targeting interferon-alpha

Effective recognition of viral infections and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs （miRNAs）. A previous study showed that miR-4661 upregulates IL-IO expression in macrophages by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation. However, the ability of miR-4661 to regulate antiviral immune responses remains unknown. Here, we found that interferon-alpha （IFN-a） expression was repressed in Sendai virus （SeV）- and vesicular stomatitis virus （VSV）-infected macrophages and in dendritic cells transfected with miR-4661 expression. Moreover, multiple IFN-α species can be directly targeted by miR-4661 through their 3＇ untranslated region （3＇UTR）. This study has demonstrated that miR-4661 could directly target IFN-a expression to inhibit host antiviral innate immune response.

α-干扰素联合索拉菲尼对肝癌细胞增殖的抑制作用及其对STAT3通路的影响

目的:探讨α-干扰素(IFN-α)联合索拉菲尼对肝癌细胞增殖的抑制作用及其对转录激活因子3(STAT3)的影响。方法:选取人肝癌细胞株HepG2,分为空白对照组(未经任何处理的HL-60细胞)、IFN-α组(加入4000 U/ml IFN-α)、索拉菲尼组(加入2μmol/L索拉菲尼)、IFN-α+索拉菲尼组(加入4000 U/ml和2μmol/L索拉菲尼),取经药物处理后的各组细胞混悬液,继续培养24、48、72 h,采用CCK8检测HepG2细胞存活率,MTT法检测HepG2细胞增殖抑制率,行蛋白质免疫印迹(Western blot)、实时定量PCR(qRT-PCR)测HepG2细胞中STAT3、Survivin mRNA和蛋白表达,并进行细胞形态学观察。结果:空白对照组细胞形态完整,轮廓清晰,呈梭形,贴壁状态良好,IFN-α组、索拉菲尼组部分胞浆肿胀、胞膜破裂、细胞形态、胞膜界限模糊不清,IFN-α+索拉菲尼组,细胞核缩小、染色质固缩、部分细胞碎裂、死亡。IFN-α+索拉菲尼组细胞增殖抑制率高于IFN-α组、索拉菲尼组(均P<0.05)。与空白对照组、IFN-α组、索拉菲尼组比较,IFN-α+索拉菲尼组细胞G 1期细胞器增多,S、G 2期细胞减少(均P<0.05)。与空白对照组比较,IFN-α组、索拉菲尼组、IFN-α+索拉菲尼组细胞中STAT3、Survivin mRNA和蛋白表达均降低,但IFN-α组和索拉菲尼组比较无统计学差异(均P<0.05),IFN-α+索拉菲尼组低于其余三组(均P<0.05)。结论:α-干扰素联合索拉菲尼可抑制HepG2细胞增殖并诱导凋亡,其机制可能是通过调节STAT3通路发挥作用。

Antitumor effects of human interferon-alpha 2b secreted by recombinant bacillus Calmette-Guérin vaccine on bladder cancer cells

Objective:Our objective was to construct a recombinant bacillus Calmette-Guérin vaccine(rBCG) that secretes human interferon-alpha 2b(IFNα-2b) and to study its immunogenicity and in vitro antitumor activity against human bladder cancer cell lines T24 and T5637.Methods:The signal sequence BCG Ag85B and the gene IFNα-2b were amplified from the genome of BCG and human peripheral blood,respectively,by polymerase chain reaction(PCR).The two genes were cloned in Escherichia coli-BCG shuttle-vector pMV261 to obtain a new recombinant plasmid pMV261-Ag85B-IFNα-2b.BCG was transformed with the recombinant plasmid by electroporation and designated rBCG-IFNα-2b.Mononuclear cells were isolated from human peripheral blood(PBMCs) and stimulated with rBCG-IFNα-2b or wild type BCG for 3 d,and then cultured with human bladder cancer cell lines T24 and T5637.Their cytotoxicities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Results:BCG was successfully transformed with the recombinant plasmid pMV261-Ag85B-IFNα-2b by electroporation and the recombinant BCG(rBCG-IFNα-2b) was capable of synthesizing and secreting cytokine IFNα-2b.PBMC proliferation was enhanced significantly by rBCG-IFNα-2b,and the cytotoxicity of PBMCs stimulated by rBCG-IFNα-2b to T24 and T5627 was significantly stronger in comparison to wild type BCG.Conclusions:A recombinant BCG,secreting human IFNα-2b(rBCG-IFNα-2b),was constructed successfully and was superior to control wild type BCG in inducing immune responses and enhancing cytotoxicity to human bladder cancer cell lines T24 and T5637.This suggests that rBCG-IFNα-2b could be a promising agent for bladder cancer patients in terms of possible reductions in both clinical dosage and side effects of BCG immunotherapy.

补肾调经汤联合人重组干扰素-α-2b对子宫内膜异位症体液免疫、CA125、EMAb水平的影响

【目的】探析补肾调经汤(在《景岳全书》大营煎方的基础上加味而成)联合人重组干扰素-α-2b治疗子宫内膜异位症(endometriosis,EMs)疗效及其对体液免疫、血清糖类抗原125(carbohydrate antigen,CA125)、抗子宫内膜抗体(antiendometrial antibody,EMAb)水平的影响。【方法】将86例EMs肾虚血瘀证患者随机分为观察组和对照组,每组各43例。对照组单纯给予人重组干扰素-α-2b治疗,观察组给予补肾调经汤联合人重组干扰素-α-2b治疗,疗程为1个月。观察2组患者治疗前后体液免疫因子[免疫球蛋白A(immunoglobulin A,IgA)、补体3(complement 3,C3)、补体4(complement 4,C4)]、子宫内膜异位包块直径及病理相关因子(CA125、EMAb)水平的变化情况,并评价2组患者的临床疗效和安全性。【结果】(1)治疗1个月后,观察组的总有效率为97.67%(42/43),对照组为81.40%(35/43),组间比较(χ2检验),观察组的疗效明显优于对照组(P<0.05)。(2)治疗后,2组患者的血清IgA水平均较治疗前明显升高(P<0.05),血清C3、C4水平均较治疗前明显降低(P<0.05),且观察组对血清IgA水平的升高幅度及对血清C3、C4水平的降低幅度均明显优于对照组(P<0.01)。(3)治疗后,2组患者的血清CA125、EMAb水平及子宫内膜异位包块直径均较治疗前明显降低(P<0.05),且观察组对血清CA125、EMAb水平及子宫内膜异位包块直径的降低幅度均明显优于对照组(P<0.01)。(4)观察组的不良反应总发生率为6.98%(3/43),对照组为11.63%(5/43),组间比较,差异无统计学意义(P>0.05)。【结论】补肾调经汤联合人重组干扰素-α-2b治疗EMs肾虚血瘀证患者临床疗效显著,可有效改善患者体液免疫因子及血清CA125、EMAb水平,缩小子宫内膜异位包块,且具有较高的安全性。