Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

重组干扰素α-2b联合CO_(2)激光气化治疗宫颈低级别上皮内病变伴HR-HPV感染患者的临床效果及对HPV转阴率的影响

目的分析重组干扰素α-2b联合CO_(2)激光气化治疗宫颈低级别上皮内病变伴高危型人乳头瘤病毒(HR-HPV)感染的临床效果。方法选取宫颈低级别上皮内病变伴HR-HPV感染患者80例,采用随机数表法分为对照组及研究组,每组40例。对照组采用重组干扰素α-2b治疗,研究组采用重组干扰素α-2b联合CO_(2)激光气化治疗。比较两组患者的治疗效果。结果研究组治疗总有效率为90.00%,高于对照组的72.50%(P<0.05)。研究组治疗3、6个月的HPV转阴率均高于对照组(P<0.05)。两组不良反应总发生率比较,差异无统计学意义(P>0.05)。结论采用重组干扰素α-2b联合CO_(2)激光气化治疗宫颈低级别上皮内病变伴HR-HPV感染患者,可提高疗效及HPV转阴率。

Study on pharmacodynamics of recombinant interferon α-2b suppository

Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the antiviral activity of this drug as well as yingtelong and axiluowei as positive control.The guinea pig model of vaginitis and skin infection caused by HSV-2 infection were established,treated with IFNα-2b suppository at dosages of 60000、180000、540000 IU,using IFNα-2b injection 180000 IU·kg-1 as controls.Score the pathological changes of appearance and skin,the virus activities of vaginal secretion and tissue sections of viginae were assayed after treatment.Results The TD50 of IFN α-2b and yingtelong for Vero cells was(>100)μg·mL-1 and(>100000)IU·mL-1,respectively.The IC50 of IFN α-2b and yingtelong and axiluowei for Herpes virus type 1 was(0.29±0.08)μg·mL-1 and(185.0±28.8)IU·mL-1 and(0.19±0.03)μg·mL-1,respectively.The mean scores for vaginal and skin lesion of the treated groups were lower than those of untreated group.Among these concentrations,the IFNα-2b suppository of 540000 IU·kg-1 group.Showed highest anti-viral activity.The virus activity in vaginal secretion of treated group was lower than that of untreated group too(P<0.01 or P<0.05).Tissue sections of viginae after treatment with IFNα-2b suppository showed significantly therapeutical effects on the degrees of vaginal lesion.At the same dosage,The anti-HSV activity of IFNα-2b suppository was also compared with IFNα-2b injection,the results showed that the activity of suppository of 540000 IU·kg-1 group was similar to that of the injection.Conclusions The IFNα-2b suppository has anti-viruses function both in vivo and in vitro.

SYNERGIC CYTOTOXICITY TO GASTRIC CANCER CELLS BY COMBINED USE OF TRICHOSANTHIN ANDRECOMBINANT INTERFERON α-2B

Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of TCS, rIFN α- 2b and their combination against digestive system cancer cell SGC- 7901.Results In the experiment in vitro, TCS, rIFN α- 2b both had direct, dose dependent cytotoxicity againstSGC - 7901. Their combined use demonstrated a toxicity signijicantly higher than that of the two drugs used alone,showing a signilicant synergic effect. This synergic cytotoxicity was confirmed in the animal experiment.Conclusion Combined use of TCS and rIFN α - 2b decreases the therapeutic dose of TCS and its toxic adverseellect, and this synergic effect is favorable to the clinical use of TCS protein against gastric cancer.

Pegylated interferon α-2b up-regulates specific CD8+ T cells in patients with chronic hepatitis B

AIM:To investigate the effect of pegylated interferon (IFN) α-2b on specific CD8+ T lymphocytes in patients with chronic hepatitis B (CHB). METHODS:Twenty-one patients with CHB were treated with pegylated IFN α-2b. Periphery blood mononuclear cells were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation (density:1.077 g/L,Pharmingen) at weeks 0,4,8,12,and 24,respectively. Frequency of circulating hepatitis B virus (HBV) epitope-specific CD8 T cells was detected by flow cytometry. Cytokines were detected by cytometric bead assay. RESULTS:The frequency of circulating HBV core or env-specific CD8 T cells was higher (P < 0.05),the number of HBV core specific CD8 T cells was greater at week 24 (P < 0.05),the level of Th1-type cytokines [interleukin (IL)-12,tumor necrosis factor-α,and IFN-γ] was higher,while that of Th2-type cytokines (IL-4,IL-6,and IL-10) was lower in responders than in nonresponders (P < 0.05) after pegylated IFN α-2b treatment. The IL-6 level was correlated with HBV DNA (r = 0.597,P = 0.04),while the inducible protein-10 (IP-10) level was correlated with serum alanine aminotransferase (ALT) (r = 0.545,P = 0.005). The IP-10 level at week 8 after pegylated IFN α-2b treatment could predict the normalization of ALT in CHB patients (positive predict value = 56%,negative predict value = 92%). CONCLUSION:Pegylated IFN α-2b can enhance the immune response of CHB patients by increasing the frequency of HBV specific CD8+ T cells and regulating the Th1/Th2 cytokines.

Expression of Human Interferon α 2b Gene in Ginseng Cells

The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.

小儿豉翘清热颗粒联合重组人干扰素喷雾剂治疗EV71感染手足口病患儿的疗效及对核因子-κB、炎症因子的影响

目的:研究小儿豉翘清热颗粒联合重组人干扰素(rhIFN)-α2b喷雾剂治疗EV71病毒感染手足口病(HFMD)患儿疗效及对核因子-κB(NF-κB)、炎症因子的影响。方法:回顾性分析我院收治的130例被EV71病毒感染的轻症HFMD患儿的临床资料及随访资料,根据治疗方法不同将患儿分为对照组和观察组各65例,对照组采用rhIFN-α2b治疗,观察组在对照组治疗基础上加用小儿豉翘清热颗粒治疗,比较两组患儿临床疗效,症状消退时间,治疗前后NF-κB、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、IL-10水平变化,肝肾功指标及药物不良反应。结果:与对照组比较,观察组临床疗效更高(P<0.05);观察组症状消退时间均短于对照组(P<0.05);与治疗前比较,两组患儿治疗后血清NF-κB、TNF-α、IL-6、IL-10水平均有所降低,且观察组低于对照组(P<0.05);两组患儿治疗前后丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、肌酐(Cr)及尿素氮(BUN)水平均为正常范围,但观察组治疗前后肝肾功能指标差值低于对照组(P<0.05);两组患儿均未发现明显药物不良反应。结论:小儿豉翘清热颗粒联合rhIFN-α2b喷雾剂治疗EV71病毒致儿童HFMD,能够有效提高治疗疗效,降低NF-κB、炎症因子,且对肝肾功能指标影响小,安全性高,值得临床推广应用。

Effects of interferon α-2b on liver function, complement level and oxidative stress in patients with hepatitis B

Objective:To observe the effect of interferonα-2b treatment on liver function,liver fibrosis,complement protein and oxidative stress in patients with hepatitis B.Methods:A total of 100 patients with hepatitis B in our hospital were randomly divided into the control group and the observation group,with 50 cases in each group.After admission,patients in the control group were treated with entecavir,while patients in the observation group were treated with interferonα-2b combined with entecavir.The levels of serum total bilirubin(TBil),aspartate aminotransferase(AST),alanine aminotransferase(ALT),type III procollagen(PCIII),type IV collagen(CIV),complement C3 protein(C3),complement C4 protein(C4),malondialdehyde(MDA),superoxide dismutase(SOD)and nitric oxide(NO)were compared between the two groups before and after treatment.Results:After treatment,the levels of TBil,AST,ALT,PCIII,CIV,MDA and NO in serum of patients with hepatitis B in both groups were significantly lower than those before treatment,and the levels of C3,C4 and SOD were significantly higher than those before treatment(P<0.05).After treatment,the levels of TBil,AST,ALT,PC III,C IV,MDA and NO in serum of patients in the observation group were significantly lower than those in the control group,while the levels of C3,C4 and SOD in serum of patients in the observation group were significantly higher than those in the control group(P<0.05).Conclusions:The combination of interferonα-2b and entecavir has a good curative effect on hepatitis B.It can significantly improve the liver function and immune function of patients,delay the process of liver fibrosis and reduce oxidative stress injury.It is worthy of clinical promotion.

Effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with CINⅢ complicated by high-risk HPV positive

Objective: To study the effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with cervical intraepithelial neoplasia (CIN) Ⅲ complicated by high-risk HPV positive. Methods: Patients who were diagnosed with CINⅢ and high-risk HPV positive and underwent conization in the 3201 Hospital Affiliated to Xi'an Jiaotong University between June 2014 and February 2017 were selected and randomly divided into the observation group who received preoperative anti-HPV bioprotein dressing combined with interferon α-2b therapy and the control group who received no special treatment. CIN lesion was collected to determine the expression of pro-proliferation molecules, pro-apoptosis molecules and epithelial-mesenchymal transition molecules. Results: Rsf1, Piwil2, TOPK, p38MAPK, ERK, Snail, Twist, N-cadherin and Vimentin mRNA expression in cervical intraepithelial neoplasia lesions of observation group were greatly lower than those of control group whereas LRIG3, SARI, IEX-1, FHIT and E-cadherin mRNA expression were greatly higher than those of control group. Conclusion: Anti-HPV bioprotein dressing combined with interferon α-2b therapy can inhibit the proliferation and invasive growth of tumor cells in patients with CINⅢ complicated by high-risk HPV positive.

自体CIK细胞联合IL-2治疗老年B细胞恶性淋巴瘤的价值探讨

目的:探讨自体细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK)联合重组人白介素-2(recombinant human interleukin-2,rh IL-2)治疗老年B细胞恶性淋巴瘤的价值。方法:对本院血液科收治的85例老年B细胞恶性淋巴瘤患者实施自体CIK细胞联合rh IL-2治疗(CIK^+IL-2组),选取同时期未接受自体CIK细胞联合rh IL-2治疗老年B细胞性恶性淋巴瘤患者85例作为对照组。CIK^+IL-2组和对照组按淋巴瘤类型分为4亚组:A组:弥漫性大B细胞淋巴瘤(DLBCL);B组:黏膜相关淋巴组织淋巴瘤(MALT);C组:淋巴浆细胞淋巴瘤(LPL);D组:霍奇金淋巴瘤(HL)。观察患者临床疗效、治疗前后T淋巴细胞亚群、β2微球蛋白水平和生存质量变化情况以及远期生存情况。结果:CIK^+IL-2组4亚组患者治疗后CD3^+、CD3^+/CD8^+、CD3^+/CD56^+水平均显著高于同组治疗前和对照组,β2微球蛋白水平均显著低于同组治疗前和对照组,差异具有统计学意义(P<0.05);CIK^+IL-2组4亚组患者经8个疗程治疗后除1例死亡外,其余CRu、PR患者疗效均转变为CR,各亚组间CR率差异无统计学意义(P>0.05);4亚组患者治疗后躯体功能、角色功能、认知功能、情绪功能及社会功能评分均显著高于同组治疗前,差异具有统计学意义(P<0.05);所有自体CIK联合IL-2治疗组患者中位生存时间为22.36±5.38(8-76)个月,对照组患者中位生存时间为(16.15±3.62(7-55)个月(P<0.05)。结论:自体CIK细胞联合rh IL-2治疗老年B细胞恶性淋巴瘤疗效显著,可有效改善患者T细胞亚群及β2微球蛋白水平,提高生存质量,延长生存时间。

拉米夫定联合重组人干扰素α-2b治疗慢性乙型病毒性肝炎的临床效果

目的 探究拉米夫定和重组人干扰素α-2b联合治疗慢性乙型病毒性肝炎的临床疗效和安全性。方法 选择2012年5月~2013年6月于我院接受治疗的86例已确诊为慢性乙型病毒性肝炎的患者作为研究对象,所有患者均给予常规的护肝治疗并随机分为实验组和对照组。其中对照组患者40例,在常规治疗的基础上给予拉米夫定进行治疗;实验组患者46例,实验组患者在此基础上联合应用重组人干扰素α-2b进行治疗,两组患者的治疗疗程均为48周,观察两组患者治疗后血常规、血清HBV复制指标和肝功能的变化及不良反应情况。结果 治疗24周后,两组患者临床指标丙氨酸氨基转移酶（ALT）复常率、乙肝病毒脱氧核糖核酸（HBV-DNA）阴转率、乙型肝炎病毒e抗原（HBeAg）阴转率、HBeAg转化率比较,差异无统计学意义（P〉0.05）;治疗48周后,以上临床指标比较,差异有统计学意义（P〈0.05）,实验组的总有效率显著高于对照组（P〈0.05）。两组患者不良反应发生率比较,差异无统计学意义（P〉0.05）。结论 拉米夫定和重组人干扰素α-2b联合治疗慢性乙型病毒性肝炎疗效显著,且优于单纯应用拉米夫定治疗,无明显不良反应,安全性可靠,值得临床推广应用。

重组人干扰素α-2b联合胸腺法新治疗慢性乙型肝炎分析

目的:探讨重组人干扰素α-2b联合胸腺法新治疗慢性乙型肝炎临床疗效。方法:分析我院收治的80例慢性乙型肝炎临床资料,依据治疗方式不同分为常规治疗组40例和联合治疗组(重组人干扰素α-2b联合胸腺法新治疗组)40例。结果:联合治疗组ALT、TBIL、ALB明显优于常规治疗组,同时联合治疗组HBVDNA病毒学转换率和HBeAg血清学转换率均高于常规治疗组,P<0.05,差异均有统计学意义。结论:重组人干扰素α-2b联合胸腺法新治疗慢性乙型肝炎患者临床效果明显,预后良好。

拉米夫定联合重组人干扰素α-2b治疗慢性乙型病毒性肝炎的临床疗效

目的探讨拉米夫定联合重组人干扰素α-2b治疗慢性乙型病毒性肝炎的临床疗效。方法选择2014年1~12月在我院治疗的慢性乙型病毒性肝炎患者60例为研究对象,随机分为研究组和对照组各30例。两组均给予拉米夫定治疗,研究组在此基础上给予重组人干扰素α-2b治疗。比较两组治疗后ALT恢复正常率、HBV-DNA转阴率、HBeAg转阴率、HBeAg转化率、总有效率以及不良反应情况。结果治疗后,研究组ALT恢复正常率、HBV-DNA转阴率、HBeAg转阴率、HBeAg转化率均显著高于对照组,差异有统计学意义(P<0.05)。研究组总有效率显著高于对照组,差异有统计学意义(P<0.05)。两组不良反应比较差异无统计学意义(P>0.05)。结论拉米联合夫定重组人干扰素α-2b治疗慢性乙型病毒性肝炎能够显著提高临床疗效,并且未明显增加不良反应,值得临床推广。

清热解毒方联合干扰素a-2b治疗宫颈高危型人乳头瘤病毒持续感染随机平行对照研究

[目的]观察清热解毒方联合干扰素a-2b治疗宫颈高危型人乳头瘤病毒(HR-HPV)持续感染疗效。[方法]使用随机平行对照方法,将68例住院患者按随机数字表法分为两组。对照组72例干扰素a-2b,隔日一粒,睡前放置于阴道后穹隆接近宫颈口处。治疗组142例清热解毒方(山药、车前子、苍术、鸡冠花、猪苓各15g,白花蛇舌草、生薏苡仁、蒲公英各30g,柴胡、芡实各10g,炒白术20g),1剂/d,水煎300mL,早晚口服,月经干净后服用,经期停药。西药治疗同对照组。连续治疗30d为1疗程。观测临床症状、血液学检查、HPV复查、不良反应。连续治疗3疗程,判定疗效。[结果]治疗组痊愈93例,有效34例,无效15例,总有效率89.44%;对照组痊愈21例,有效39例,无效12例,总有效率83.33%。转阴93例,转阴率65.49%。临床疗效治疗组优于对照组(P<0.05)。治疗组HR-HPV病毒转阴率优于对照组(P<0.01)。[结论]清热解毒方联合干扰素a-2b治疗HR-HPV持续感染效果显著,值得推广使用。

重组人干扰素α-2b联合拉米夫定治疗慢性乙型肝炎120例疗效观察

目的研究重组人干扰素α-2b(安达芬)联合拉米夫定(LAM)对慢性乙型肝炎患者的疗效。方法将120例慢性乙型肝炎患者按1:1比例随机分为2组:联合组给予重组人干扰素α-2b和LAM治疗;单用组给予LAM治疗,疗程均为1年。结果治疗1年后,联合组血清HBV-DNA转阴率和HBeAg/抗HBe转换率均高于单用组(P均<0.05),单用组YMDD变异率高于联合组(P均<0.05)。结论重组人干扰素α-2b(安达芬)联合拉米夫定(LAM)治疗的1年疗效优于单用拉米夫定。

rhIL-12联合HBsAg刺激健康人外周血单核细胞对IL-12R的影响

目的确认重组人白细胞介素12(rhIL-12)联合乙型肝炎表面抗原(HBsAg)可增强健康人外周血单核细胞(PBMC)产生特异性细胞免疫应答,并分析其细胞来源,及对IL-12R的影响,探讨hIL-12联合HBsAg作为治疗型乙型肝炎疫苗的应用前景。方法 8名健康人PBMC分别与rhIL-12(1 ng/mL)、rHBsAg(500 ng/mL)和rhIL-12(1ng/mL)+rHBsAg(500 ng/mL)体外孵育,采用流式细胞术检测CD4+IFN-γ(γ-干扰素)+T细胞、CD8+IFN-γ+T细胞及CD56+IFN-γ+NK细胞百分比,并测定表达IL-12Rβ1、IL-12Rβ2的细胞百分比。结果 rhIL-12与rHBsAg联合组CD8+IFN-γ+T细胞及CD56+IFN-γ+NK细胞百分比均较对照组明显升高,且有非常显著差异(P<0.01),联合组CD8+IL-12Rβ1和CD56+IL-12Rβ1与单用rhIL-12或rHBsAg组比较均显著升高。结论提示rhIL-12联合rHBsAg诱生的IFN-γ主要来自CD56+NK细胞、CD8+T细胞,其次是CD4+T细胞;rhIL-12和rHBsAg单独均能上调健康人PBMCs表面IL-12R的表达,rhIL-12与rHBsAg联合具有增强作用。

Bcl-2、Bad在大鼠癫痫持续状态中的表达及rHuEpo干预的分子机制

目的观察PTZ致痫的SD大鼠SE时海马神经元损伤的情况,给予rHuEpo干预后的变化以及应用PI3K抑制剂LY294002后神经元凋亡以及Bcl-2、Bad的变化情况,对rHuEpo的作用机制进行更深一步的探讨。方法采用PTZ致痫大鼠SE模型,随机将大鼠分为正常对照组、模型组、rHuEpo干预组、LY294002干预组、LY294002对照组,用TUNEL法检测海马神经元凋亡状况;免疫组化SP法观察p-Akt、Bcl-2、Bad阳性细胞的表达;每组大鼠海马Bcl-2 mRNA、Bad mRNA的表达采用RT-PCR方法检测,每组大鼠海马p-Akt、Bcl-2、Bad蛋白的表达采用Western blot方法检测。结果 rHuEpo干预后可以上调p-Akt、Bcl-2 mRNA和Bcl-2蛋白的表达、减少Bad mRNA和Bad蛋白的表达,对神经元具有一定的保护作用;与rHuEpo干预组相比,PI3K抑制剂LY294002干预后,pAkt、Bcl-2 mRNA和蛋白的表达明显减少,海马Bad mRNA和蛋白的表达较rHuEpo干预组明显增加,rHuEpo的神经保护作用被明显减弱,差异具有统计学意义。结论从正反两个方面提示rHuEpo的抗凋亡作用可能通过对PI3K/Akt信号通路发挥影响,从而对凋亡线粒体途径中Bcl-2、Bad蛋白的表达程度产生调节,最终实现对抗癫痫持续状态凋亡损伤的神经保护作用。

重组人α-2b干扰素含片药效学试验

目的观察重组人α-2b干扰素（rhIFN α-2b）舍片对体外、体内的抗病毒作用，为临床用药提供药理学依据。方法采用体外和体内抗病毒试验。结果rhIFN α-2b含片浓度为400～250000IU／mL时，对单纯疱疹病毒（HSV-1、HSV2）和水痘-带状疱疹病毒有明显抑制作用，并在31．2—1000IU／mL浓度时，对乙型肝炎病毒也有明显抑制作用。小鼠抗流感病毒试验表明，口含rhIFN α-2b 500～50000IU／只，能明显降低动物死亡率，并延长存活时间。结论rhIFN α-2h含片对体内、体外病毒均有明显的抑制作用。