Expression of Interleukin-15 and Its Receptor on the Surface of Stimulated Human Umbilical Vein Endothelial Cells

Summary： Human interleukin-15 （hIL-15） is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ （IFN-γ）, and tumor necrosis factor-or （TNF-α to induce the production of human interleukin-15 （hIL-15） and IL-15 receptor （IL-15Rα by human umbilical vein endothelial cells （HUVECs）. The data are summarized as follows： 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting （FACS）, and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-α and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-α and TNF-α play an important role in regulating the expression of IL-15 and IL-15Rα on the surface of HUVECs.

Modulation of Interleukin-15-induced Suppression of Human Neutrophil Apoptosis by TNFα

Human interleukin-15 （IL-15） is a proinflammatory cytokine to suppress neutrophil apoptosis, which is a potential therapeutic agent. The modulatory effect of TNFα was investigated in IL-15-induced suppression of human neutrophil apoptosis. TNFa was shown to reverse the ability of IL-15 to delay neutrophil apoptosis within certain time course. Moreover, this reverse effect by TNFα might be associated with a reduction of the expression of the anti-apoptotic Bcl-XI protein detected by Western blotting. It is concluded that TNFα can be used to modulate IL- 15-induced suppression of neutrophil apoptosis within certain time course.

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.

Involvement of interleukin-15 and interleukin-21, two γ-chain-related cytokines, in celiac disease

Celiac disease (CD), an enteropathy caused by dietary gluten in genetically susceptible individuals, is histologically characterized by villous atrophy, crypt cell hyperplasia, and increased number of intra-epithelial lymphocytes. The nature of CD pathogenesis remains unclear, but recent evidence indicates that both innate and adaptive immune responses are necessary for the phenotypic expression and pathologic changes characteristic of CD. Extensive studies of molecules produced by immune cells in the gut of CD patients have led to identification of two cytokines, namely interleukin (IL)-15 and IL-21, which are thought to play a major role in orchestrating the mucosal inflammatory response in CD. Here we review the current knowledge of the expression and function of IL-15 and IL-21 in CD.

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

Human IL15 cDNA fragment, which contains all codons encoding the human IL15 mature protein and signal peptide was transducted into the human lung squmouse cancer cells(PG cells) and murine lung adenocarcinoma cells(LA795 cell lines). Two IL15 highly expressed cell clones PG1 and LA795A were used to inoculate the nude mice and the T739 syngeneic mice respectively. PG1 cell express higher level of class ⅠMHC molecule on their surface than PG cells. It was shown that the modified LA795A tumor cells grew slowly in T739 mice and induced high levels of CTL/NK/LAK activity in vivo as well, compared with the case of inoculation with LA795 or LA795neo. No significant difference in the tumor growth was observed in groups of the nude mice inoculated by PG1, PG and PGneo cells respectively, except the gene modified cells could not show the lung metastasis of tumors. The supernatants derived from the LA795A cell culture could promote CTL/NK/LAK activity from the whole splenocytes and the CD4/CD8deleted splenic cells in vitro. The results indicated that the IL15 gene transfected tumor cells play important roles in the process of antitumor or antitumor metastasis.

Polymorphisms in interleukin-10 gene according to mutations of NOD2/CARD15 gene and relation to phenotype in Spanish patients with Crohn's disease

AIM： To examine the contribution of interleukin-10 （IL-10） gene polymorphisms to Crohn＇s disease （CD） phenotype, and the possible genetic epistasis between IL-10 gene polymorphisms and CARD15/NOD2 gene mutations. METHODS： A cohort of 205 Spanish unrelated patients with Crohn＇s disease recruited from a single center was studied. All patients were rigorously phenotyped and followed-up for at least 3 years （mean time, 12.5 years）. The clinical phenotype was established prior to genotyping. RESULTS： The correlation of genotype-Vienna classification groups showed that the Ueocolonic location was significantly associated with the -1082G allele in the NOD2/CARD15 mutation-positive patients （RR = 1.52, 95%CI, 1.21 to 1.91,P= 0.008）. The multivariate analysis demonstrated that the IL-10 G14 microsatellite allele in the NOD2/CARD15 mutation positive patients was associated with two risk factors, history of appendectomy （RR = 2.15, 95%CI = 1.1-4.30, P= 0.001） and smoking habit at diagnosis （RR= 1.29, 95%CI= 1.04-4.3, P= 0.04）. CONCLUSION： In Spanish population from Madrid, in CD patients carrying at least one NOD2/CARD15 mutation, the -1082G allele is assodated with ileocolonic disease and the IL-IOG14 microsatellite allele is associated with previous history of appendectomy and smoking habit at diagnosis. These data provide further molecular evidence for a genetic basis of the clinical heterogeneity of CD.

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.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Recombinant human interleukin-11 for treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer

Objective： To evaluate the efficacy and safety of recombinant human interleukin-11 （rhIL-11） for the chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer. Methods： It was an opened and non-randomized controlled clinical study. When the platelet counts was under 75 × 10^9/L after chemotherapy, rhlL-11 was administered 25 μg/（kg·d） as a daily SC injection last for 7-14 days, or discontinued when platelet counts 〉 100 × 10^9/L. Results： Seventysix patients were enrolled into this study. The treatment group and the control group had thirty-eight cases, respectively. The mean recovery time to PLT ≥ 100 × 10^9/L was 8.1 days in treatment group, while in control group was 12.2 days （P 〈 0.01）. Moreover, the mean recovery time from PLT 〈_ 50 × 10^9/L to 〉 100 × 10^9/L was 8.9 days in treatment group, while in control group was 12.9 days （P 〈 0.05）. There was a statistical difference between the two groups. Major side effects included edema, fever, articular muscle soreness, but they were all mild and well tolerable. Conclusion： rhIL-11 can be safely and effectively used for the treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer.

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Upregulation of stromal cell-derived factor-1 alpha/CXCR4 axis-induced migration of human neural progenitors by tumor necrosis factor-alpha and interleukin-8

BACKGROUND： Studies of several animal models of central nervous system diseases have shown that neural progenitor cells （NPCs） can migrate to injured tissues. Stromal cell-derived factor 1 alpha （SDF-la）, and its primary physiological receptor CXCR4, have been shown to contribute to this process. OBJECTIVE： To investigate migration efficacy of human NPCs toward a SDF-1α gradient, and the regulatory roles of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） in SDF-1α/CXCR4 axis-induced migration of NPCs. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, cellular and molecular biology study was performed at the Laboratory of Department of Cell Biology, Medical College of Soochow University between October 2005 and November 2007. MATERIALS： SDF-1α and mouse anti-human CXCR4 fusion antibody were purchased from R＆D Systems, USA. TNF-αwas purchased from Biomyx Technology, USA and IL-8 was kindly provided by the Biotechnology Research Institute of Soochow University. METHODS： NPCs isolated from forebrain tissue of 9 to 10-week-old human fetuses were cultured in vitro. The cells were incubated with 0, 20, and 40 ng/mL TNF-α, or 0, 20, and 40 ng/mL IL-8, for 48 hours prior to migration assay. For antibody-blocking experiments, cells were further pretreated with 0, 20, and 40 μg/mL mouse anti-human CXCR4 fusion antibody for 2 hours. Subsequently, the transwell assay and CXCR4 blockade experiments were performed to evaluate migration of human NPCs toward a SDF-1α gradient. Serum-free culture medium without SDF-1α served as the negative control. MAIN OUTCOME MEASURES： The transwell assay was performed to evaluate migration of human NPCs toward a SDF-1α gradient, which was blocked by fusion antibody against CXCR4. In addition, CXCR4 expression in human NPCs stimulated by TNF-α and IL-8 was measured by flow cytometry. RESULTS： Results from the transwell assay demonstrated that SDF-1α was a strong chemoattractant for human NPCs （P 〈 0.01）, and 20 ng/mL produced the highest levels of migration. Anti-human CXCR4 fusion antibody significantly blocked the chemotactic effect （P 〈 0.05）. Flow cytometry results showed that treatment with TNF-α and IL-8 resulted in increased CXCR4 expression and greater chemotaxis efficiency of NPCs towards SDF-1α（P 〈 0.01）. CONCLUSION： These results demonstrated that SDF-la significantly attracted NPCs in vitro, and neutralizing anti-CXCR4 antibody could block part of this chemotactic function. TNF-α and IL-8 increased chemotaxis efficiency of NPCs towards the SDF-1αgradient by upregulating CXCR4 expression in NPCs.

RESPONSES OF HUMAN FETAL SPLENOCYTES AND THYMOCYTES TO INTERLEUKIN-2: LAK ACTIVITY AND PROLIFERATION

Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.

人脐带间充质干细胞通过GDF-15/FOXO3a治疗小鼠卵巢功能不全的研究

目的:探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)改善卵巢功能不全(premature ovarian insufficiency,POI)小鼠的卵巢损伤及机制。方法:采用足底注射透明带3多肽(zona pellucida3 peptide,pZP3)方法构建POI小鼠模型,将小鼠分为对照组、佐剂对照组、pZP3组和hUC-MSCs组,每组10只。以阴道涂片监测动情周期、以酶联免疫吸附测定检测血清卵泡刺激素(follicle-stimulating hormone,FSH)和雌二醇(estradiol,E_(2))水平,以HE染色观察卵巢组织学,以蛋白质印迹(Western blotting)检测叉头框转录因子O3a(forkhead box O3a,FOXO3a)、p-FOXO3a、p53、胱天蛋白酶-3(Caspase-3)、Bax表达水平,以实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)检测POI的标志物骨形态发生蛋白15(bone morp hogenetic protein 15,BMP15)、抗米勒管激素(anti-Müllerian hormone,AMH)、WNT、卵泡刺激素受体(follicle-stimulating hormone receptor,FSHR)以及促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和IL-1β的表达水平,以RNA-seq检测生长分化因子-15(growth differentiation factor-15,GDF-15)的表达水平。通过环磷酰胺(cyclophosphamide,Cy)构建体外模型。结果:(1)造模6周后,与对照组相比,pZP3组和hUC-MSCs组动情周期出现紊乱,且血清FSH水平升高,血清E_(2)水平降低,表明模型构建成功。(2)相较于对照组,pZP3组闭锁卵泡增加,原始卵泡数目降低,hUC-MSCs干预后小鼠原始卵泡比例和数目均高于pZP3组(P<0.05)。(3)与对照组相比,pZP3组卵巢中凋亡蛋白p53、Bax表达水平上调,hUC-MSCs干预后小鼠凋亡蛋白p53、Bax表达较pZP3组下调(均P<0.05);pZP3组体内Caspase-3变化与对照组差异无统计学意义(P>0.05)。pZP3组炎症因子IL-1β、TNF-α表达上调,hUC-MSCs干预小鼠的IL-1β、TNF-α表达较pZP3组显著下调(均P<0.05)。(4)pZP3组体内Treg细胞数量降低(P<0.01),hUC-MSCs干预后小鼠Treg细胞数量较pZP3组升高(P<0.0001)。pZP3组卵巢组织中BMP15、AMH、WNT以及FSHR的mRNA表达水平较对照组降低,hUC-MSCs干预后小鼠的表达水平较pZP3组升高(均P<0.05)。(5)pZP3组FOXO3a磷酸化水平高于对照组(P<0.0001),hUC-MSCs干预后,FOXO3a磷酸化水平较pZP3组下降(P<0.0001)。测序结果提示,pZP3组GDF-15表达上调,hUC-MSCs组中GDF-15表达较pZP3组下调。结论:POI小鼠体内GDF-15和p-FOXO3a表达上调,h UC-MSCs通过下调GDF-15的表达和降低FOXO3a的磷酸化水平,改善POI小鼠的卵巢组织形态,实现对POI小鼠的治疗作用。

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

血清人附睾蛋白4、糖类抗原15-3、ROMA指数对上皮性卵巢癌复发的预测价值

目的分析血清人附睾蛋白4(HE4)、糖类抗原15-3(CA15-3)联合卵巢癌风险评估模型(ROMA)指数对上皮性卵巢癌(EOC)复发的预测价值。方法选取2018年1月1日至2022年1月1日于该院妇产科就诊的135例EOC患者作为研究对象。分析HE4、CA15-3水平及ROMA指数在不同肿瘤类型、淋巴结转移情况、国际妇产联盟分期(FIGO)分期及复发情况的EOC患者上的差异。绘制受试者工作特征(ROC)曲线分析HE4、CA15-3及ROMA指数单独及3项指标联合检测对EOC患者3年复发的预测价值。结果浆液性囊腺癌患者的ROMA指数、HE4、CA15-3水平均高于黏液性囊腺癌、子宫内膜样腺癌、交界性癌、透明细胞癌、其他类型癌患者,差异均有统计学意义(P<0.05);子宫内膜样腺癌患者HE4水平明显高于黏液性囊腺癌、交界性癌、透明细胞癌及其他类型癌患者,且透明细胞癌患者HE4水平明显高于黏液性囊腺癌、交界性癌及其他类型癌患者,差异均有统计学意义(P<0.05);透明细胞癌患者CA15-3水平明显高于黏液性囊腺癌、子宫内膜样腺癌、交界性癌及其他类型癌患者,且子宫内膜样腺癌患者CA15-3水平明显高于黏液性囊腺癌、交界性癌及其他类型癌患者,差异均有统计学意义(P<0.05);子宫内膜样腺癌患者ROMA指数明显高于黏液性囊腺癌、交界性癌、透明细胞癌及其他类型癌患者,且透明细胞癌患者ROMA指数明显高于黏液性囊腺癌、交界性癌及其他类型癌患者,差异均有统计学意义(P<0.05)。FIGO分期Ⅲ期患者ROMA指数、HE4、CA15-3水平均明显高于FIGO分期Ⅰ期及FIGO分期Ⅱ期患者,且FIGOⅡ期患者明显高于FIGOⅠ期患者,差异均有统计学意义(P<0.05)。有淋巴结转移的患者ROMA指数、HE4、CA15-3水平均明显高于无淋巴结转移的患者,差异均有统计学意义(P<0.05)。在规定的时间内,共计有28例患者出现复发,107例患者未出现复发。EOC复发患者的ROMA指数、HE4、CA15-3水平均明显高于未复发EOC患者,差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,HE4、CA15-3、ROMA指数单独及3项指标联合预测ECO患者3年内复发的曲线下面积(AUC)分别为0.670、0.716、0.669及0.798。结论EOC患者血清HE4、CA15-3水平及ROMA指数与患者的术后复发密切相关,术前联合检测血清HE4、CA15-3水平及ROMA指数有助于进一步提高对EOC患者术后复发的预测价值。

血清HE4、CA125、CA15-3水平联合检测在卵巢肿瘤良恶性诊断中的效能

目的:分析人附睾蛋白4(HE4)、癌胚抗原125(CA125)、糖类抗原15-3(CA15-3)水平联合检测在卵巢肿瘤良恶性诊断中的效能。方法:选取2022年1月至2023年6月该院收治的78例卵巢肿瘤患者进行横断面研究,依据病理学检查结果将其分为卵巢癌组48例和卵巢良性病变组30例,另选取同期30名体检的健康女性设为对照组。比较三组、不同分期卵巢癌组患者血清HE4、CA125、CA15-3水平,绘制受试者工作特征(ROC)曲线分析HE4、CA125、CA15-3水平单项及联合检测在卵巢肿瘤良恶性诊断中的效能。结果:卵巢癌组HE4、CA125、CA15-3水平均高于卵巢良性病变组、对照组,卵巢良性病变组CA125水平高于对照组,差异有统计学意义(P<0.05);卵巢良性病变组与对照组HE4、CA15-3水平比较,差异均无统计学意义(P>0.05);Ⅲ~Ⅳ期卵巢癌患者HE4、CA125、CA15-3水平均高于Ⅰ~Ⅱ期患者,差异有统计学意义(P<0.05);ROC曲线分析结果显示,HE4、CA125、CA15-3水平单项及联合检测诊断卵巢癌的曲线下面积分别为0.731、0.695、0.636、0.953,且联合检测诊断效能高于三者单项检测诊断效能。结论:血清HE4、CA125、CA15-3水平联合检测诊断卵巢肿瘤良恶性的效能高于三者单项检测诊断。

LncRNA TPTEP1/miR-137/KLF15轴在肥胖相关性肾病中的作用机制研究

目的:探讨长链非编码核糖核酸(LncRNA)人肌力蛋白磷酸酶同源假基因1(TPTEP1)/微小核糖核酸-137(miR-137)/Kruppel样因子15(KLF15)轴在肥胖相关性肾病(ORKD)中的作用机制。方法:选择赣南医科大学第一附属医院2022年2月至2024年1月20例ORKD住院者为ORKD组,同期住院的非ORKD患者20例为对照组,采集血液进行生化检验,通过免疫组化及电镜检查肾活检标本病理变化,利用生物信息学工具对比分析ORKD组织与正常肾组织中KLF15信使核糖核酸(mRNA)的表达水平,酶联免疫吸附试验分析血清脂联素、瘦素水平。结果:ORKD组患者的血肌酐(Scr)、尿酸(UA)、24 h尿蛋白定量(24h-UTP)水平高于对照组,差异均具有统计学意义(P<0.05);ORKD组肾组织KLF15表达水平、血清脂联素水平相比于对照组明显降低,瘦素水平明显增加,差异均具有统计学意义(P<0.05);高通量测序筛查LncRNA发现,ORKD组ENSG00000100181.15表达差异大,miR-137可能是LncRNA TPTEP1的靶基因,LncRNA TPTEP1在机体多种组织中均有表达,其中包括肾脏及脂肪组织。结论:ORKD的肾组织中KLF15和脂联素的异常可能与疾病的发展有关,LncRNA TPTEP1/miR-137/KLF15轴在ORKD发展过程中发挥着重要的作用。

三种IL-15基因转染NCI-H446细胞模型的建立与鉴定

目的构建并鉴定三种IL-15基因转染的人小细胞肺癌(NCI-H446)细胞模型。方法PCR法扩增得原型IL-15基因片段(FhIL-15)I、L-15成熟肽基因片段(FhIL-15mp)和IL-2信号肽基因片段(FhIL-2sp);利用重叠延伸基因拼接法将FhIL-15mp和FhIL-2sp拼接成改型IL-15基因片段(FhIL-2sp-hIL-15mp)。将三种IL-15基因片段(FhIL-15、FhIL-15mp和FhIL-2sp-hIL-15mp)分别插入真核表达载体pEGFP-N1构建成相应的重组质粒(PhIL-15、PhIL-15mp和PhIL-2sp-hIL-15mp),用脂质体法分别转染NCI-H446、G418筛选得三种IL-15基因转染的NCI-H446细胞(C-hIL-15、C-hIL-15mp、C-hIL-2sp-hIL-15mp)。对所得的各基因片段和重组质粒,用琼脂糖凝胶电泳和测序鉴定;对所得的各转染细胞,用RT-PCR法检测hIL-15mRNA的表达,ELISA法检测hIL-15蛋白的分泌。结果琼脂糖凝胶电泳和测序证明,FhIL-15、FhIL-15mp、FhIL-2sp-hIL-15mp电泳条带位置和基因序列正确。三种转染细胞均有IL-15mRNA表达,但仅C-hIL-2sp-hIL-15mp可测到22pg/mL的IL-15蛋白分泌。初步应用表明,三种转染细胞确有不同的免疫生物学特性。结论正确构建了三种IL-15基因转染的NCI-H446细胞模型,可在进一步研究IL-15基因与肿瘤细胞免疫生物学特性的关系及至机制中应用。

rhIL-15表达载体的构建及其在大肠杆菌中的表达

目的 获得rhIL 15基因工程菌。方法 从肺癌细胞株A549中提取细胞总RNA ,用RT PCR法扩增编码人IL 15成熟区基因的片段。经EcoRⅠ和XhoⅠ双酶切后 ,插入融合表达质粒pGEX 4T 2的相应酶切位点 ,构建重组融合蛋白表达质粒 ,并转化感受态大肠杆菌BL2 1而得到工程菌。结果 重组质粒插入片段核酸序列测定的结果 ,与文献报道的IL 15蛋白成熟区的核酸序列相一致。该工程菌所表达的融合蛋白多数以包涵体的形式存在 ,占菌体蛋白总量的 39%。复性后 ,纯化的rhIL 15蛋白经MTT比色法初步测定 ,具有促进CTLL 2细胞增殖的能力。结论 获得了具有生物学活性的rhIL

重组人白细胞介素15表达载体的构建及其在大肠杆菌中的高效表达

目的 :获得重组人白细胞介素 1 5 (rhIL 1 5 )高效表达菌株。方法 :经细菌脂多糖 +γ干扰素活化的人外周血单个核细胞提取细胞总RNA ,用RT PCR方法扩增出编码人IL 1 5cDNA的基因片段 ,采用 pBV2 2 0表达载体 ,经DNA重组技术构建IL 1 5基因工程菌。结果 :核酸序列测定与预期一致 ,所表达的IL 1 5经SDS PAGE证明分子量约 1 5kD ,表达量占菌体总蛋白的 2 8% ,经CTLl2 细胞检测 ,表达产物粗提物 1∶1 0 0复性后效价可达到1 0 6IU /ml。结论 :构建的基因工程菌为IL 1 5高效表达菌株。