Expression,Purification and Identification of Recombinant Mouse Interleukin 21 Protein in E.coli

Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .

Expression, purification and bioactivity of human augmenter of liver regeneration

AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase （GST） sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS： The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups （P 〈 0.01）.CONCLUSION： Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.

Expression and Purification of SARS Coronavirus Membrane Protein

To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E.Coli) BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.9921 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

EXPRESSION OF NITROREDUCTASE GENE NOR_1 IN E.Coli AND THE PREPARATION OF ANTISERUM

Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.

Expression of GST-IL-1 fusion gene

Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ,SDS-PAGE was employed to detect the gene expression. No corresponding protein encoded by GST gene fused with the whole-length 816 bp IL-1 cDNA was observed, nor was free GST protein. However, the fusion protein of GST and IL-1 cDNA without the 189 bp at the 5'- terminus was detected, amounting to 30% of the total bacterial protein expressed. This might suggest that the sequence of 1-189 bp of IL-1 cDNA affected the expression of the fusion gene. That is to say, the downstream sequence distant from the translation start codon AUG in the target gene could significantly affect the expression of the fusion gene.

老年舒张性心力衰竭合并肌少症患者可溶性生长刺激表达基因2蛋白、肌红蛋白、白细胞介素-6水平与心功能的相关性

目的 探讨老年舒张性心力衰竭(DHF)合并肌少症患者外周血可溶性生长刺激表达基因2蛋白(sST2)、肌红蛋白(Myo)、白细胞介素-6(IL-6)水平与心功能的相关性。方法 将122例DHF患者根据有无肌少症分为DHF合并肌少症组60例和DHF组62例,另将健康体检者58例、单纯肌少症患者60例分别纳入对照组、单纯肌少症组,检测各组外周血sST2、Myo、IL-6水平和心功能指标[左室射血分数(LVEF)、心排血量(CO)、心率(HR)、每搏输出量(SV)和心脏指数(CI)]。采用Pearson相关分析法分析sST2、Myo、IL-6与各心功能指标的相关性。绘制受试者工作特征(ROC)曲线,分析sST2、Myo、IL-6单独及联合诊断DHF合并肌少症的效能。结果 与对照组、单纯肌少症组相比,DHF组、DHF合并肌少症组sST2、Myo、IL-6水平和HR均升高,LVEF、CO、SV和CI均降低,差异有统计学意义(P<0.05);与DHF组相比,DHF合并肌少症组sST2、Myo、IL-6水平和HR均升高,LVEF、CO、SV和CI均降低,差异有统计学意义(P<0.05)。sST2、Myo、IL-6均分别与LVEF、CO、SV、CI呈负相关(P<0.001),均与HR呈正相关(P<0.001);sST2、Myo、IL-6、LVEF、SV是DHF合并肌少症的独立影响因素(P<0.05);sST2、Myo、IL-6联合诊断DHF合并肌少症的曲线下面积为0.936,诊断效能优于三者单独检测。结论 老年DHF合并肌少症患者外周血sST2、Myo、IL-6水平显著升高,且sST2、Myo、IL-6均与心功能指标显著相关,三者联合检测对DHF合并肌少症的诊断效能较高。

血清IL-2、sST2表达与特发性膜性肾病免疫抑制剂治疗反应性的相关性

目的探讨特发性膜性肾病患者血清白介素-2(IL-2)、可溶性生长刺激表达基因2蛋白(sST2)表达水平与免疫抑制剂治疗反应性的相关性。方法选取2020年1月至2022年10月于医院接受免疫抑制剂治疗的135例特发性膜性肾病患者,于入院时检测患者血清IL-2、sST2,并于治疗完成后测定24 h尿蛋白定量,依据患者治疗反应性分为缓解组与未缓解组。对比两组患者一般资料及入院时血清IL-2、sST2水平,采用点二列相关性分析血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性的关系,并绘制受试者工作特征(ROC)曲线评估血清IL-2、sST2水平预测特发性膜性肾病免疫抑制剂治疗反应性的价值。结果135例特发性膜性肾病患者中共有132例完成规律治疗,经免疫抑制剂治疗6个月后,101例患者疾病缓解,纳入缓解组,其余31例患者纳入未缓解组。未缓解组年龄、入院时肾功能分级、疾病分期、血清IL-2、sST2水平均高于缓解组,差异有统计学意义(P<0.05);点二列相关性分析显示,血清IL-2、sST2水平与特发性膜性肾病免疫抑制剂治疗反应性不良风险呈正相关(r 1=0.428,P 1<0.001;r 2=0.344,P 2<0.001);绘制ROC曲线,结果显示,血清IL-2、sST2预测特发性膜性肾病免疫抑制剂治疗反应性不良的曲线下面积均>0.7,具有一定预测价值,且联合预测价值更高。结论血清IL-2、sST2表达水平与特发性膜性肾病患者免疫抑制剂治疗反应性密切相关,二者表达水平越高,治疗反应性越差,且联合检测可作为预测特发性膜性肾病患者免疫抑制剂治疗反应性的敏感指标。

猪繁殖与呼吸综合征病毒GP5蛋白在昆虫杆状病毒表达系统中的表达与鉴定

猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)GP5蛋白是病毒主要囊膜结构蛋白和中和抗体主要靶蛋白,广泛应用于PRRS新型疫苗和诊断技术的研究。为获得具有与天然构象相近的PRRSV GP5蛋白,本研究根据草地贪夜蛾(Spdopterafrugiperda)密码子偏好性,优化PRRSV ORF5基因编码密码子,并克隆至杆状病毒载体后转染Sf9昆虫细胞,构建了重组杆状病毒rBac-PRRSV-GP5。经Western blot证实GP5蛋白在Sf9细胞中获得高效表达,且具有良好的免疫反应原性。经镍离子亲和层析纯化后,获得高纯度的重组GP5蛋白,为开展PRRSV诊断技术研发、生物学功能研究等奠定了抗原基础。

IL-21编码基因的克隆及其在大肠杆菌中表达

①目的 利用基因工程技术从人扁桃体细胞cDNA中克隆出白细胞介素 2 1(IL 2 1)的编码基因 ,并在大肠杆菌中进行表达。②方法 以人扁桃体细胞经PHA刺激的cDNA文库为模板 ,经PCR获得IL 2 1的全基因片段。测序确认后 ,分别设计含BamHⅠ和SalⅠ酶切位点的引物 ,经PCR获得IL 2 1成熟肽的编码基因。将此IL 2 1基因插入表达载体 pGEX4T 2 ,重组克隆经酶切与测序确认后转化大肠杆菌DH5α进行IPTG诱导表达。 ③结果 琼脂糖凝胶电泳显示重组质粒的PCR和双酶切产物与理论值的大小相等。SDS PAGE显示经IPTG诱导后的大肠杆菌DH5α亦表达一与理论相符的条带。目的蛋白约占总菌体蛋白的 10 %。④结论 成功地构建了IL 2

胃癌相关基因GCRG213在大肠杆菌中的表达及重组蛋白纯化

目的 利用硫氧还蛋白融合表达系统表达胃癌相关基因GCRG2 13,制备高纯度的GCRG2 13蛋白。方法 采用PCR技术从pGEM T质粒上扩增出含完整ORF的GCRG2 13cDNA序列 ,将其克隆至硫氧还蛋白融合表达载体 pET10 2 /D TOPO中 ,转化大肠杆菌BL2 1,经IPTG诱导表达融合蛋白 ,凝胶回收目的蛋白。结果 SDS PAGE证实在大肠杆菌中高效表达出相对分子量约 2 9 4kD的Thioredoxin/GCRG2 13融合蛋白。薄层凝胶扫描显示 ,其表达量占菌体总蛋白的 2 8 7%。经凝胶回收法得到纯度近 10 0 %的蛋白产品。结论 在大肠杆菌中成功表达了Thioredoxin/GCRG2 13融合蛋白 ,并制备出高纯度蛋白产品 。

人白细胞介素21基因重组复制缺陷型腺病毒的制备与鉴定

目的制备表达绿色荧光蛋白的含人白细胞介素21(IL-21)基因的重组复制缺陷型腺病毒,为IL-21基因治疗肿瘤的研究奠定基础。方法从人外周血淋巴细胞提取总RNA,经RT-PCR扩增IL-21基因片段,PCR产物和pDC316-mCMV-EGFP载体分别经限制性内切酶NotI与HindIII双酶切,然后将酶切产物连接、转化,构建pDC316-IL21-EGFP重组质粒。重组质粒经酶切和测序鉴定后,与腺病毒骨架质粒pBHGlox_E1,3CreF35共转染293细胞,包装产生Ad5F35-IL21-EGFP重组腺病毒,并测定病毒滴度。观察Ad5F35-IL21-EGFP腺病毒转染后食管癌细胞EC-9706的生长曲线和细胞周期的变化。结果 pDC316-IL21-EGFP重组质粒经酶切和测序证实,IL-21基因片段正确插入pDC316-mCMV-EGFP载体中且与GenBank中IL-21基因序列一致。Ad5F35-IL21-EGFP重组腺病毒载体在293细胞中包装成功,获得成熟的病毒颗粒,经鉴定有IL-21基因和绿色荧光蛋白的表达。测得Ad5F35-IL21-EGFP病毒颗粒滴度为9×1011VP/ml,感染性滴度为5×1010IU/ml。Ad5F35-IL21-EGFP转染后,EC-9706细胞的生长缓慢(P<0.05),G1期细胞增加,而G2期和S期细胞减少。结论成功制备了表达绿色荧光蛋白的人IL-21基因的重组腺病毒,且病毒滴度较高。腺病毒介导的外源性IL-21基因可有效转染EC-9706细胞,并对其生长有抑制作用。

成纤维细胞生长因子21在大鼠各组织中表达分布的研究

目的:在基因及蛋白水平探讨成纤维细胞生长因子21(Fibroblast growth factor 21,FGF21)是否在大鼠各组织中表达及其表达水平,为进一步研究FGF21在各组织器官中的作用提供依据。方法:对大鼠肝脏、脂肪、心脏、主动脉、肾脏及肌肉等各组织中FGF21含量进行Real Time-PCR及放射免疫定量检测,在基因及蛋白水平检测FGF21在大鼠各组织器官中的表达情况,并比较其在各组织器官中的表达水平。结果:Real Time-PCR及放射免疫测定结果显示,在大鼠肝脏、脂肪、心脏、主动脉、肾脏及肌肉等各组织中均有FGF21 mRNA及其蛋白表达,并存在一定差异。同时各组织中基因及蛋白水平的表达量并不完全平行[△Ct:(1.068±0.1682),(9.165±1.784),(11.91±0.3394),(9.410±1.650),(11.90±0.1931),(12.74±0.5176),P<0.01];蛋白:[(0.0160±0.005066),(0.0700±0.01728),(0.01514±0.004451),(0.03671±0.005992),(0.2077±0.03045),(0.01017±0.006616),P<0.01]。结论:FGF21在大鼠各主要组织器官中均有表达,并存在一定差异。

结核分枝杆菌培养滤液蛋白21(CFP21)的基因克隆、表达及纯化

目的构建结核分枝杆菌培养滤液蛋白21(Culture filtrate protein 21,CFP21)原核表达载体,获得CFP21蛋白。方法以结核分枝杆菌H37Rv株基因组DNA为模板,PCR扩增cfp21基因,质粒PET28a(+)为表达载体,构建PET28a(+)/cfp21质粒,转化入大肠埃希菌DH5α中,抽提质粒,PCR扩增,测序,转化到大肠埃希菌BL21中,用IPTG诱导表达,SDS-PAGE分析,并用His Bind蛋白纯化试剂盒纯化CFP21。结果构建、表达和纯化了CFP21,分子量约为24kD,主要以包涵体形式存在。结论目的基因克隆入宿主菌中并表达成功,纯化得到CFP21蛋白,为CFP21的进一步研究奠定基础。

海洋黄杆菌来源岩藻多糖酶Fcn1的异源表达及酶学性质

为了寻找和挖掘降解岩藻多糖的新型酶,以从海带中筛选获得的一株可以降解岩藻多糖的海洋黄杆菌Flavobacterium sp.RC2-3为研究对象,对其进行了全基因组测序。通过与已报道的岩藻多糖酶氨基酸序列比对,并进行转录组学分析及实时荧光定量PCR验证,挖掘了1个可能的岩藻多糖酶基因,并将其命名为Fcn1。Fcn1基因全长1221 bp,编码406个氨基酸,蛋白分子质量约为46.8 kDa。通过引物设计、PCR扩增,将Fcn1基因克隆,进一步构建了Fcn1基因异源表达载体Fcn1-pET-28a(+),并将其成功地在大肠杆菌BL21(DE3)表达宿主中进行了诱导表达。所得重组酶Fcn1通过带有His标签的镍柱进行分离纯化,采用铁氰化钾法测定纯化酶酶解岩藻多糖的比酶活(332 U/mg),纯化倍数为2.25。酶学性质研究表明,重组酶Fcn1最适反应温度为50℃,最适反应pH值为8.0,在20~30℃和pH值为7.0~8.0时表现出较好的稳定性。结合酶学性质研究结果,确定酶的最适反应条件为30℃,pH值为8.0,并测定了岩藻多糖酶水解反应的动力学参数,K m为1.17 mg/mL,V_(max)为10.53 g·L^(-1)·min^(-1)。该酶降解岩藻多糖的酶活力较高,在岩藻多糖资源的开发领域具有较大应用潜力。

牛种布鲁氏菌ArsR2蛋白的原核表达及磷酸化位点鉴定

用原核表达系统表达并纯化牛种布鲁氏菌S2308株ArsR2蛋白,软件分析该蛋白相关信息,质谱鉴定该蛋白修饰位点。以牛种布鲁氏菌S2308株基因组为模板,通过PCR扩增arsR2基因,构建pET-32a-ArsR2重组质粒,经测序正确后,转化至大肠埃希氏菌感受态细胞BL21(DE3)中进行IPTG诱导表达,通过Ni-NTA亲和层析法进行ArsR2蛋白的纯化,并进行SDS-PAGE和Western blot鉴定;对牛种布鲁氏菌ArsR2蛋白的理化性质和磷酸化位点进行预测,通过质谱鉴定ArsR2蛋白的修饰位点。结果显示,成功表达并纯化ArsR2蛋白,ArsR2蛋白无跨膜区,存在磷酸化修饰,质谱鉴定出ArsR2蛋白具有2个磷酸化位点。表明原核表达的牛种布鲁氏菌S2308株ArsR2蛋白存在磷酸化修饰位点,为探究该蛋白的生物学功能奠定了基础。

屋尘螨过敏原Der p 1在毕赤酵母中的重组表达与纯化

基于巴斯德毕赤酵母表达系统探讨了屋尘螨过敏原Der p 1的重组表达与纯化。首先,采用毕赤酵母GS115表达密码子优化的全长编码基因PreProDer p 1,其产量可达100 mg/L,进一步共表达分子伴侣实现产量提高到140 mg/L,并实现3 L反应器发酵产量提高到1 g/L。其次,对上述重组蛋白发酵液分别使用阳离子交换层析及亲和层析进行了纯化工艺优化,目的蛋白得率达到60.7%。本研究为后续PreProDre p 1诊断试剂盆的开发提供了参考。

STX 5对肝细胞癌转移的影响及其机制

目的探讨STX5对肝细胞癌(HCC)转移的影响及其机制。方法收集2015年1—12月我院确诊为HCC的36例患者的临床资料,分析肿瘤组织中STX5表达水平与HCC患者临床病理特征的相关性。将人肝癌细胞MHCC97H分为A、B组,分别转染阴性对照质粒、过表达STX5质粒,将人肝癌细胞Huh7分为C、D组,分别转染阴性对照慢病毒、STX5敲减慢病毒,采用Western blot实验检测A~D组细胞内STX5蛋白表达水平,采用划痕实验检测A~D组细胞的迁移能力,采用Transwell实验检测A~D组细胞的迁移能力。对A、B组细胞经转录组学测序分析获得的差异基因进行GO功能和KEGG信号通路富集分析,实时荧光定量聚合酶链反应(RT-qPCR)检测A、B组细胞最显著差异表达基因的水平。将MHCC97H细胞分为E~H组,分别转染阴性对照质粒、过表达STX5质粒、阴性对照质粒+Sarilumab、过表达STX5质粒+Sarilumab,采用划痕实验检测E~H组细胞的迁移能力,采用Transwell实验检测E~H组细胞的迁移能力。结果肿瘤组织中STX5表达水平与患者的BMI、有无乙型肝炎病毒感染、肿瘤数量有关(P<0.05)。Western blot检测结果显示,B组与A组、C组与D组比较,细胞中STX5的表达显著增高(t=48.86、31.09,P<0.05)。Transwell实验和划痕实验结果显示,B组与A组、C组与D组比较,细胞迁移能力和划痕愈合百分比显著升高(t=7.95~31.09,P<0.05)。GO和KEGG富集分析显示,A、B组细胞差异基因主要富集在细胞迁移、炎症等相关功能和通路上;RT-qPCR实验结果显示,B组细胞中IL-6 mRNA的表达水平显著高于A组细胞(t=23.69,P<0.05)。Transwell和划痕实验结果显示,G组和E组、H组和F组比较,细胞迁移能力和划痕愈合百分比均显著降低(t=2.94~24.39,P<0.05)。结论STX 5可能通过上调IL-6 mRNA表达来促进肝细胞癌细胞的转移。