Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.

Expression and significance of intratumoral interleukin-12 and interleukin-18 in human gastric carcinoma

AIM: To explore the effect of intratumoral expressions of interleukin-12 (IL-12) and interleukin-18 (IL-18) on clinical features, angiogenesis and prognosis of gastric carcinoma. METHODS: The expressions of IL-12 and IL-18 from 50 samples of gastric cancer tissue were analyzed by immunohistochemistry, and microvessel density (MVD) was determined with microscopic imaging analysis system. RESULTS: The positive expression rates of IL-12 and IL-18 were 44% (22/50) and 26% (13/50), respectively. IL-12 was significantly associated with pathologic differentiation, depth of invasion, lymph node metastasis, distant metastasis, and TNM stage, and IL-18 was closely related to distant metastasis. Intratumoral IL-12 and IL-18 expressions were not statistically related to MVD scoring. IL-12-positive patients survived significantly longer than those with IL-12-negative tumors, but there was no significant difference between IL-18-positive patients and IL-18-negative ones. The multivariate analysis with Cox proportional hazard model revealed IL-12, MVD and T stage were independent prognostic factors. CONCLUSION: The positive expressions of IL-12 and IL-18 can play an important role in progression and metastasis of gastric cancer, and IL-12 might be an independent factor of poor prognosis in gastric carcinoma.

Study on interleukin-18 gene transfer into human breast cancer cells to prevent tumorigenicity

To study the effect of interleukin-18 gene transfection on the tumorigenesis of breast cancer cell line Bacp37,human breast cancer cell line Bcap37 were transfected with Lipofectamine and selected by G418.The biological expression of rhIL-18 was tested by RT-PCR and ELISA method;nude mice were injected with Bcap37 cell with or without the hIL-18 gene.The hIL-18 cDNA was successfully integrated into Bcap37 cell; 126.3±4.5pg hIL-18 secreted by one million transduced cells in 24 hours. Nude mice injected with IL-18 gene engineered Bcap37 cell had no tumor growth.These findings indicated that human breast cancer cells were successfully modified by the gene of IL-18 cytokine;the IL-18 gene engineered Bcap37 cells secreted hIL-18 and lost their tumorigenicity.The Bcap37 cells transduced with IL-18 gene may be used as breast cancer vaccine.

Analysis of the Role of D-Dimer,Interleukin-6,and Interleukin-18 in Differential Diagnosis of Pediatric Refractory Mycoplasma pneumoniae Pneumonia

Objective:To analyze the value of D-dimer(D-D),interleukin-6(IL-6),and IL-18 in the differential diagnosis of children with refractory Mycoplasma pneumoniae pneumonia(RMPP).Methods:The medical records of 92 children with Mycoplasma pneumoniae pneumonia(MPP)treated in the hospital were selected for retrospective analysis from January 2023 to January 2024.After comprehensive examinations such as computed tomography examination of the chest,48 children with general Mycoplasma pneumoniae pneumonia(GMPP)were put in the GMPP group and 44 children with RMPP were grouped in the RMPP group.The IL-6,IL-18,and D-D levels were compared between the two groups,and the receiver operating characteristic(ROC)curves were plotted to analyze their value for differential diagnosis of RMPP.Results:The levels of IL-6,IL-18,and D-D in the RMPP group were higher than those in the GMPP group(P<0.05);the ROC curves showed that the specificity of the differential diagnosis of IL-6,IL-18,and D-D was higher,and their diagnostic value was significant.Conclusion:Determination of IL-6,IL-18,and D-D levels in children with MPP can further diagnose the children’s condition,which can help physicians formulate targeted treatment plans,and is of great significance to the improvement of the children’s condition,which is worthy of attention.

血清中Interleukin-18含量的检测对结肠癌预后判断的价值

目的 探讨结肠癌患者血清中白细胞介素 18(IL 18)水平与预后的关系。方法 收集 10 6例结肠癌患者和 60例健康成人的血清 ,采用酶联免疫吸附反应 (ELISA )检测IL 18的水平。结果 1997年 6月及以前的血清 5 9份 ,IL 18的平均含量为3 3 8.46pg/ml ;1997年 6月以后的血清 47份 ,IL 18的平均含量为 3 2 8.85 pg/ml ;2组比较无显著性差异 (P <0 .0 5 ) ;结肠癌组患者血清IL 18的含量 ( 3 46.5 3 pg/ml± 3 1.2 3pg/ml)明显高于正常对照组 ( 2 3 3 .3 2 pg/ml± 2 2 .5 4pg/ml) ,有显著性差异 (P <0 .0 5 ) ;Ⅱ期、Ⅲ期和Ⅳ期患者血清中IL 18水平明显高于对照组 (P <0 .0 5 ) ,血清IL 18≥ 3 46pg/ml组的患者 5年生存率为 5 .3 % ,血清IL 18<3 46pg/ml的患者 5年生存率为 18.6% ;两组比较有显著性差异 (P <0 .0 5 ) ;多因素分析表明 ,血清IL 18含量是判断结肠癌患者预后的独立因素。结论 血清中IL 18含量与结肠癌患者 5年生存率有密切相关。血清中IL

ILTV Glycoprotein B(gB)与鸡Interleukin-18(IL-18)真核双表达载体的构建及表达

将ILTVgB基因和鸡IL-18基因插入到真核双表达载体pIRES中,构建共表达gB和IL-18基因的重组质粒pIRES-gB/IL18和表达gB基因的pIRES-gB质粒。通过脂质体将其转染鸡胚成纤维细胞,利用RT-PCR及间接免疫荧光检测重组质粒在体外的表达。将重组质粒通过腿部肌肉多点注射免疫21日龄雏鸡,应用ELISA和流式细胞仪分别检测免疫鸡的体液免疫及细胞免疫水平。结果显示,pIRES-gB/IL18和pIRES-gB转染细胞中分别含gB/IL-18基因的mRNA和gB基因的mRNA。间接免疫荧光检测表明,pIRES-gB/IL18和pIRES-gB在CEF细胞中有效地转录并表达gB蛋白。pIRES-gB/IL18和pIRES-gB免疫雏鸡后均能促进外周血T淋巴细胞亚群数量和血清中特异性抗体水平的增加,但前者的免疫效果明显优于后者,表明gB基因和鸡IL-18基因均获得了表达,且鸡IL-18能明显增强ILTV DNA疫苗诱发的免疫应答。

卵巢癌血清CCL18、HK10水平变化及与肿瘤恶性生物学行为的关系

目的探讨卵巢癌血清趋化因子18(CCL18)、人激肽释放酶10(HK10)水平变化及与肿瘤恶性生物学行为的关系。方法选取2018年1月至2023年4月河北省沧州中西医结合医院收治的100例卵巢癌患者作为研究组,50例良性卵巢肿瘤患者作为对照组,50名健康志愿者作为健康组。比较三组血清CCL18、HK10水平,研究组不同肿瘤恶性生物学行为患者血清CCL18、HK10水平,研究组不同化疗灵敏度患者化疗前和化疗3、6个周期后血清CCL18、HK10水平变化值,分析化疗前后血清CCL18、HK10水平变化值与化疗灵敏度的关系及对化疗灵敏度的预测价值。结果研究组血清CCL18、HK10水平均高于对照组、健康组(P<0.05)。研究组不同国际妇产科联盟(FIGO)分期、淋巴结转移、肌层浸润程度血清CCL18和HK10水平、不同分化程度血清HK10水平比较,差异具有统计学意义(P<0.05)。研究组化疗不灵敏患者CCL18△1(△1为化疗3个周期后与化疗前变化值的绝对值)、HK10△1均低于化疗灵敏患者(P<0.05)。CCL18△1、HK10△1联合检测的曲线下面积(AUC)显著高于单一指标(P<0.05)。结论卵巢癌血清CCL18、HK10水平升高与肿瘤恶性生物学行为关系密切,且其变化值能够辅助临床预测化疗灵敏度,两者联合检测具有较高的化疗灵敏度预测价值。

Association of polymorphisms of interleukin-18 gene promoter region with chronic hepatitis B in Chinese Han population

AIM: To investigate the polymorphisms of interleukin-18 (IL-18) gene promoters, and to disclose whether such polymorphisms are associated with susceptibility to chronic hepatitis B in Chinese Han population. METHODS: Using polymerase chain reaction with sequence specific primers (PCR-SSP) method, the single nucleotide polymorphisms (SNPs) of the promoter region of IL-18 gene at position -607 and -137 were detected in 231 patients with chronic hepatitis B and 300 normal controls. RESULTS: Allele C at position -607 in the promoter of IL-18 gene was detected in 48.7% of normal controls and 51.9% of patients, while allele A at position -607 was detected in 51.3% of normal controls and 48.1% of patients. The frequencies of -607CC, -607 CA and -607AA genotypes in normal controls were 22.0%, 53.3% and 24.7% respectively and in chronic hepatitis B patients were 26.8%, 50.2% and 23.0% respectively. Allele G at position -137 in the promoter of IL-18 gene was detected in 82.3% of normal controls and 88.5% of chronic hepatitis B patients, while allele C at position -137 was detected in 17.7% of normal controls and 11.5% of patients. The frequencies of -137GG, GC and CC genotype were 67.3%, 30.0% and 2.7% in normal controls respectively, while in chronic hepatitis B patients were 78.8%, 19.5% and 1.7% respectively. The frequency of-137GG genotype in chronic hepatitis B groups was significantly higher than that in normal controls (x2=8.55, P=0.003 <0.05), whereas the frequencies of -607C/-137C and -607A/-137C haplotypes in chronic hepatitis B groups were significantly lower than that in normal controls. The association between genotypes of IL-18 promoter region polymorphisms and HBV copies showed that the frequency of -607AA genotype in high HBV-DNA copies groups was lower than that in low HBV-DNA copies groups (x2=6.03, P=0.014 <0.05). CONCLUSION: The polymorphisms of the promoter region of IL-18 gene at position -607 and -137 are closely associated with susceptibility to chronic hepatitis B. The people with allele C at position -137 in the promoter of IL-18 gene may be protected against HBV infection; moreover AA genotype at position -607 may be closely linked to inhibit HBV-DNA replication. These findings give some new clues to the study of pathogenesis of chronic hepatitis B.

Role of serum interleukin-18 as a prognostic factor in patients with hepatocellular carcinoma

AIM:To determine whether serum interleukin-18 (IL-18) levels correlated with clinicopathologic features and prognosis in patients with hepatocellular carcinoma (HCC). METHODS:Serum IL-18,IL-6 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) from 70 patients with HCC and 10 healthy controls. RESULTS:Serum IL-18,IL-6 and IL-12 levels of patients with HCC were significantly higher that those of the controls. The levels of IL-18 correlated significantly with the presence of venous invasion and advanced tumor stages classified by Okuda's criteria. Patients with high serum IL-18 levels (≥ 105 pg/mL) had a poorer survival than those with low serum IL-18 levels (< 105 pg/mL) (4 and 11 mo,respectively,P = 0.015). Multivariate analyses showed that serum IL-18 level,but not IL-6 and IL-12 levels,was a significant and independent prognostic factor of survival. CONCLUSION:These findings demonstrate that serum IL-8 may a useful biological marker of tumor invasiveness and an independent prognostic factor of survival for patients with HCC. Thus,the detailed mechanisms of IL-18 involving in tumor progression should be further investigated.

Plasma interleukin-18 reflects severity of ulcerative colitis

AIM: The aim of this study was to evaluate the association between ulcerative colitis activity and plasma or mucosal concentrations of interleukin (IL)-18. METHODS: 11-18 concentrations were measured in plasma and mucosal samples from 15 patients with active ulcerative colitis (UC). RESULTS: The mean plasma concentration of IL-18 measured in all patients (422±88 pg/mL) doubled the mean value in healthy controls (206±32 pg/mL); however, the difference was not statistically significant. Plasma IL-18 levels revealed a significant positive correlation with scored endoscopic degree of mucosal injury, disease activity index, clinical activity index and C-reactive protein concentration. The mean concentration of plasma IL-18 was significantly higher in patients with severe ulcerative colitis (535±115 pg/mL) than in patients with mild ulcerative colitis (195±41 pg/mL), and in healthy controls. Although the mucosal mean IL-18 concentration in severe ulcerative colitis (2 523±618 pg/mg protein) doubled values observed in mild one (1347±308 pg/mg protein), there was no statistically significant difference. CONCLUSION: Plasma IL-18 can be considered as a surrogate marker helpful in evaluation of ulcerative colitis activity.

Interleukin-18 genetic polymorphisms contribute differentially to the susceptibility to Crohn's disease

AIM:To investigate the correlation between interleukin-18(IL-18) gene polymorphisms and the risk of developing Crohn's disease(CD).METHODS:The PubMed,CISCOM,CINAHL,Web of Science,EBSCO,Cochrane Library,MEDLINE,EMBASE and CBM databases were searched without any language restrictions using combinations of keywords relating to CD and IL-18 for relevant articles published before November 1st,2013.Screening of the published studies retrieved from searches was based on our stringent inclusion and exclusion criteria and resulted in seven eligible studies for meta-analysis.A metaanalysis was conducted using a random-effects model with STATA 12.0 software.Crude odds ratios(ORs) with95%confidence intervals(95%CI) were calculated.RESULTS:Seven case-control studies,with a total of1930 CD cases and 1930 healthy subjects,met our inclusion criteria.The results of our meta-analysis indicated that the IL-18 rs1946518 A>C and rs187238G>C polymorphisms may correlate with an increased risk of CD under five genetic models(all P < 0.05).Furthermore,we observed positive associations between the IL-18 rs360718 A>C polymorphism and CD risk under three genetic models(C allele vs A allele:OR = 2.03,95%CI:1.20-3.43,P = 0.008;CC vs AA+AC:OR = 2.39,95%CI:1.2-4.43,P = 0.006;CC vs AC:OR = 2.31,95%CI:1.22-4.38,P = 0.010).However,such associations were not found for the IL-18 rs917997 C>T,codon 35 A>C and rs1946519G>T polymorphisms(all P> 0.05).A subgroup analysis was conducted to investigate the effect of ethnicity on an individual's susceptibility to CD.Our results revealed positive correlations between IL-18 genetic polymorphisms and an increased risk of CD among Asians and Africans(all P < 0.05),but not among Caucasians(all P> 0.05).CONCLUSION:This meta-analysis indicated that the IL-18 rs1946518 A> C,rs187238 G>C and rs360718A>C polymorphisms may contribute to susceptibility to CD,especially among Asians and Africans.These polymorphisms are known to reduce IL-18 mRNA and protein levels.

Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients

AIM To evaluate the treatment effects of recombinant human interleukin-12(rh IL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc.METHODS The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy(IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rh IL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervenedwith rh IL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS Rh IL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period.CONCLUSION Rh IL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

Objective:To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines.Methods:BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA;splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro.Results:The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone,but there was not significantly different (P>0.05).Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-γ in supernatant of splenocytes cultured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05).Conclusion:The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

Inhibition of hepatic interleukin-18 production by rosiglitazone in a rat model of nonalcoholic fatty liver disease

AIM:To investigate the effects of rosiglitazone(RGZ) on expression of interleukin-18(IL-18) and caspase-1 in liver of non-alcoholic fatty liver disease(NAFLD) rats.METHODS:Twenty-eight Sprague-Dawley(SD) rats were randomly divided into control,NAFLD,and RGZ treated NAFLD groups.A NAFLD rat model of NAFLD was established by feeding the animals with a high-fat diet for 12 wk.The NAFLD animals were treated with RGZ or vehicle for the last 4 wk(week 9-12) and then sacrificed to obtain liver tissues.Histological changes were analyzed with HE,oil red O and Masson's trichrome staining.Expressions of IL-18 and caspase-1 were detected using immunohistochemical staining and semi-quantitative reverse-transcription polymerase chain reaction(RT-PCR) analysis.RESULTS:The expression levels of both IL-18 and caspase-1 were higher in the liver of NAFLD group than in the control group.Steatosis,inflammation and fibrosis,found in the liver of NAFLD rats,were significantly improved 4 wk after RGZ treatment.The elevated hepatic IL-18 and caspase-1 expressions in NAFLD group were also significantly attenuated after RGZ treatment.CONCLUSION:RGZ treatment can ameliorate increased hepatic IL-18 production and histological changes in liver of NAFLD rats.The beneficial effects of RGZ on NAFLD may be partly due to its inhibitory effect on hepatic IL-18 production.

Enhanced expression of interleukin-18 in serum and pancreas of patients with chronic pancreatitis

AIM： To investigate interleukin-18 （IL-18） in patients with chronic panreatitis （CP）. METHODS： We studied 29 patients with CP and 30 healthy controls. Peripheral blood mononuclear cells （PBMC） were isolated and incubated with 50 mmol/L ethanol, lipopolysaccharide （LPS） （doses 25 g/L, 250 g/L, 2500 g/L） and both agents for 24 h. Levels of IL-18 in the supernatants, and levels of IL-18, IL-12, interferon （IFN）-T and soluble CD14 in the serum were analysed by EI_ISA technique. Expression of IL-18 in PBMC was investigated by reverse-transcription （RT）-PCR. IL-18 protein levels in CP tissue and in normal pancreas were studied by ELISA technique. IL-18 levels in PBMC and pancreatic tissue were determined by Westernblot. Immunohistochemistry for pancreatic IL-18 expression was performed.RESULTS： In patients, IL-18 serum levels were significantly enhanced by 76% （mean： 289.9 ± 167.7 ng/L） compared with controls （mean： 165.2 ± 43.6 ng/L; P 〈 0.0005）. IL-12 levels were enhanced by 25% in patients （18.3 ± 7.3 ng/L） compared with controls （14.7 ± 6.8 ng/L, P = 0.0576） although not reaching the statistical significance. IFN-γ, and soluble CD14 levels were not increased. In vitro, LPS stimulated significantly and dosedependently IL-18 secretion from PBMC. Incubation with ethanol reduced LPS-stimulated IL-18 secretion by about 50%. The mRNA expression of IL-18 in PBMC and the response of PBMC to ethanol and LPS was similar in CP patients and controls. In PBMC, no significant differences in IL-18 protein levels were detected between patients and controls. IL-18 protein levels were increased in CP tissues compared to normal pancreatic tissues. IL-18 was expressed by pancreatic acinar cells and by infiltrating inflammatory cells within the pancreas. CONCLUSION： IL-18 originates from the chronically inflammed pancreas and appears to be involved in the fibrotic destruction of the organ.

18β-glycyrrhetinic Acid-induced Apoptosis and Relation with Intracellular Ca^2＋ Release in Human Breast Carcinoma Cells

Objective:To study the effects of 18β-glycyrrhetinic acid (GA) on proliferation inhibition, apop totic induction, and the relationship between GA-induced apoptosis and intracellular Ca2+ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 μmol/L to 250 μmol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 μmol/L GA for 24 h, intracellular Ca2+ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 μmol/L to 250 μmol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose dependent fashion. IC50 of the proliferation inhibition was 234.33 μmol/L. Treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca2+ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18β-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca2+ level may be depended on apoptosis induced by GA in MCF-7 cells.

Relationship of Serum Interleukin-18 and Interleukin-12 Levels with Clinicopathology in Renal Cell Carcinoma

Objective： To investigate the relationship between serum interleukin-18 and interleukin-12 levels and clinicopathology of renal cell carcinoma. Methods： Peripheral blood samples were obtained from 20 healthy volunteers and 60 patients with renal cell carcinoma before curative surgery. IL-12 and IL-18 levels were determined by enzyme-linked immunosorbent assay. Results： Mean serum IL-12 and IL-18 levels were significantly higher in patients with renal cell carcinoma compared with healthy volunteers （P〈0.05） and mean serum IL-12 and IL-18 levels increased in patients as the pathologic stage progressed. A positive correlation was observed between serum IL-12 and IL-18 levels （P〈0.05）. In patients with renal cell carcinoma, increasing serum IL-12 and IL-18 levels correlated with pathological stage and Fuhrman grade. Conclusion： Serum IL-12 and IL-18 might be useful tumor markers in patients with renal cell carcinoma.

Effect of glycyrrhizic acid and 18β-glycyrrhetinic acid on the differentiation of human umbilical cord-mesenchymal stem cells into hepatocytes

BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options.Cell-based therapies using mesenchymal stem cells(MSCs)emerged as an alternative approach to support hepatic regeneration.In vitro preconditioning strategies have been employed to strengthen the regenerative and differentiation potential of MSCs towards hepatic lineage.Chemical compounds of the triterpene class;glycyrrhizic acid(GA)and 18β-glycyrrhetinic acid(GT)possess diverse therapeutic properties including hepatoprotection and anti-fibrosis characteristics.They are capable of modulating several signaling pathways that are crucial in hepatic regeneration.Preconditioning with hepato-protective triterpenes may stimulate MSC fate transition towards hepatocytes.AIM To explore the effect of GA and GT on hepatic differentiation of human umbilical cord-MSCs(hUC-MSCs).METHODS hUC-MSCs were isolated and characterized phenotypically by flow cytometry and immunocytochemistry for the expression of MSC-associated surface molecules.Isolated cells were treated with GA,GT,and their combination for 24 h and then analyzed at three time points;day 7,14,and 21.qRT-PCR was performed for the expression of hepatic genes.Expression of hepatic proteins was analyzed by immunocytochemistry at day 21.Periodic acid Schiff staining was performed to determine the functional ability of treated cells.RESULTS The fusiform-shaped morphology of MSCs in the treatment groups in comparison with the untreated control,eventually progressed towards the polygonal morphology of hepatocytes with the passage of time.The temporal transcriptional profile of preconditioned MSCs displayed significant expression of hepatic genes with increasing time of differentiation.Preconditioned cells showed positive expression of hepatocyte-specific proteins.The results were further corroborated by positive periodic acid Schiff staining,indicating the presence of glycogen in their cytoplasm.Moreover,bi-nucleated cells,which is the typical feature of hepatocytes,were also seen in the preconditioned cells.CONCLUSION Preconditioning with glycyrrhizic acid,18β-glycyrrhetinic acid and their combination,successfully differentiates hUC-MSCs into hepatic-like cells.These MSCs may serve as a better therapeutic option for degenerative liver diseases in future.

Interleukin-18 Suppresses Angiogenesis and Lymphangiogenesis in Implanted Lewis Lung Cancer

Objective： To investigate the effects of interleukin-18 （IL-18） on implanted Lewis lung cancer in suppressing angiogenesis and lymphangiogenesis. Methods： One week after hypodermic inoculation of Lewis cells, sixteen tumor-bearing syngeneic mice were randomly divided into two groups. The mice in the treatment group （group A） were intraperitoneally injected with the IL-18 and in the control group （group B） were intraperitoneally injected with normal saline for 7 days. The mice were killed on day 19. The morphology of the tumors was studied, the growth curve was described and the tumor inhibition rate was calculated. The numbers of metastasized pulmonary colonies were calculated using hematoxylin-eosin （HE） staining. The vascular endothelial growth factor A （VEGF-A）, vascular endothelial growth factor C （VEGF-C）, microvessel density （MVD） and lymphatic microvessel density （LMVD） in tumor mass were measured by immunohistochemistry staining （IHCS）. Results： In the group A, the tumor volume, tumor weigh, lung metastatic nodules, expression of VEGF-A and VEGF-C, MVD were all decreased significantly （P0.01 or P0.05）. The tumor inhibition rate was 75% and the inhibition rate of lung metastatic nodules was 61%. The LMVD in group A was also lower than group B, but without significant difference （P0.05）. Conclusion： IL-18 could suppress the tumor growth and metastasis of implanted Lewis lung cancer by way of down-regulating VEGF-A and VEGF-C expression, suppressing angiogenesis and lymphangiogenesis.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.