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.

Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

AIM： To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy. METHODS： Nuclear matrix proteins were selectively extracted from MGcS0-3 cells treated with or without hexamethylamine bisacetamide （HMBA）, and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ ionization time of flight mass spectrometry （MALDI-TOFMS） analysis and submitted for database searching using Mascot tool. RESULTS： The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGcS0-3 cells compared to control. Eleven of which were identified. Seven proteinsactin, prohibitin, porin 31HL, heterogeneous nuclear dbonucleoprotein A2/B1, vimentin, ATP synthase, and heat shock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated, and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells. CONCLUSION： The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

SPECIFIC BINDING OF HUMAN BONEMORPHOGENETIC PROTEIN （2A） WITH MOUSEOSTEOBLASTIC CELLS

Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM21. Human BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the surface of mouse osteoblastic cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with 3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the surface of MC3T3 cells exist the receptor for hBMP2A.

Survival of transplanted neurotrophin-3 expressing human neural stem cells and motor function in a rat model of spinal cord injury

BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.

Targeting tight junctions during epithelial to mesenchymal transition in human pancreatic cancer

Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and-18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin(CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1,-7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition(EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.

Effects of histamine on growth and apoptosis of human melanoma cells A375

Objective: To investigate the effects of histamine on growth and apoptosis of human melanoma cells A375. Methods: The effect of histamine on growth of A375 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis by double staining with Annexin V-FITC and PI, and active caspase-3 analysis by staining FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody were made by flow cytometer. StreptAvidin-Biotin Complex (SABC) immunocytochemical assays were adopted to detect Bax/Bcl-2 protein expressions.Results: Histamine inhibited proliferation of A375 cells in a dose- and time-dependent manner, and altered cell cycle distribution of A375 cells revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. Histamine induced apoptosis of A375 cells (P<0.05), elevated the cells population with detectable active caspase-3 (P<0.05), increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly (P<0.05). Conclusion: That histamine inhibits cell cycle progress of A375 cells is one of the possible mechanisms of proliferation arrest of A375 cells elicited by histamine. Histamine mediates apoptosis in A375 cells that may be caspase-dependent through mitochondria routine. Histamine with high concentration inhibits growth of A375 cells in vitro by interfering proliferation and inducing apoptosis of cells.

Effects of Exogenous Growth Hormone on Growth Hormone-Insulin-Like Growth Factor Axis of Human Gastric Cancer Cell

Aim: To study effects of recombinant human growth hormone (rhGH) on growth hormone-insulin-like growth factor axis (GH-IGFs) of human gastric cancer cell in vivo in order to reveal part mechanism of growth effects of rhGH on gastric cancer. Methods: Nude mice were randomly divided into control group, cisplatin (DDP) group, rhGH group and DDP + rhGH group after human gastric cancer xenograft model of node mice was successfully founded and drugs were used for 6 days. We investigated volume of tumor, inhibitory rate of tumor and cell cycle by slide gauge and flow cytometry. In addition, We also respectively investigated insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) of blood serum of nude mice, IGF-ImRNA, insulin-like growth factor-I receptor (IGF-IR) mRNA and IGFBP-3 mRNA of xenograft of nude mice by enzyme linked immunosorbent assay (ELISA) and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on the first day of completing use of drugs later. Results: Tumor grew obviously slowly and tumor inhibitory rate obviously rose in DDP group and DDP + rhGH group compared with control group and rhGH group (p p p < 0.05). Expressions of IGF-I mRNA and IGF-IR mRNA were not obviously different in all groups. But expression of IGFBP-3 mRNA obviously increased in rhGH group, DDP group and DDP + rhGH group compared with control group;meanwhile, expression of IGFBP-3 mRNA also obviously increased in DDP + rhGH group compared with control group, DDP group and rhGH group. Conclusion: Our results indicated rhGH in short-time use did not improve proliferation of human gastric cancer cells and its mechanism was possible that rhGH in short-time use raised simultaneously IGF-I and IGFBP-3 of blood serum and increased IGFBP-3 mRNA, but degraded ratio of IGF-I and IGFBP-3 of blood serum in human gastric cancer cells.

Signal transducers and activators of transcription 3 mediates up-regulation of angiotensin ll-induced tissue inhibitor of metalloproteinase-1 expression in cultured human senescent fibroblasts

Backgroud Angiotensin Ⅱ （Ang Ⅱ）, a principal effector of renin-angiotensin system （RAS） and increased in aging tissues, can stimulate JAK/STAT pathway via the G-protein-coupled Ang Ⅱ receptor type Ⅰ （AT1） and induce nuclear translocation of signal transducers and activators of transcription （STAT）. To further explore the role of Ang Ⅱ in aging, we examined the effect of Ang Ⅱ on human replicative senescent diploid fibroblast WI-38 cells.

The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells

AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.

Correlation between neuronal injury and Caspase-3 after focal ischemia in human hippocampus

Background Cerebral ischemia is a significant clinical problem, and cerebral ischemia usually causes neuron injury such as apoptosis in various brain areas, including hippocampus. Cysteinyl aspartate-specific protease (Caspases) are fundamental factors of apoptotic mechanism. Caspase-3 inhibitors show effect in attenuating brain injury after ischemia. But all the results were from animal models in research laboratories. This study aimed at investigating the correlation between the change of ischemic neuronal injury and Caspase-3 post-ischemia in human hippocampus. Methods We selected and systematized 48 post-mortem specimens from 48 patients, who died of cerebral infarction. Morphological change was firstly analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of Caspase-3 was investigated using the methods of in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick-end labeling (TUNEL) method was used to clarify the involvement of Caspase-3 in neuron death. The loss of MAP 2 (MAP-2) was applied to judging the damaged area and degree of neuronal injury caused by ischemia.Results In the CA1 sector of hippocampus, Caspase-3 immunostaining modestly increased at 8 hours [8.05/high-power field (hpf)], dramatically increased at 24 hours (24.85/hpf), decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6.75/hpf), reached a maximum at 16 hours (17.60/hpf), faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10.76/hpf), markedly increased at 48-72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the Caspase-3 evolution was related with the upregulation of TUNEL and the loss of MAP-2. The positive correlation between Caspase-3 mRNA and TUNEL was significant at the 0.05 level (correlation coefficient was 0.721); the negative correlation between Caspase-3 mRNA and MAP-2 was significant at the 0.05 level (correlation coefficient is 0.857). In the early stage (before 72 hours), the staining of Caspase-3 mRNA and immunohistochemistry was predominantly present in cytoplasm; the staining of TUNEL was predominantly localized in nucleus. At 4-16 hours, most neurons in hippocampal CA1 areas had relatively normal morphology; at 24-48 hours, neurons showed apoptotic morphology; at 72 hours, most cells showed significantly pathological morphology. Conclusions There exist a time-dependent evolution of neuronal damage after hippocampal ischemia in human brain, which was characterized by its close correspondence to Caspase-3.

Antitumor immunity of human SART3 gene vaccine against mouse tumor in vitro/vivo

To determine whether squamous cell carcin-oma antigen recognized by human T cell 3(SART3)gene can induce antitumor immunity against tumor cells which express the gene,we constructed mouse tumor cells expressing human SART3(LM8-SART3)and carried out experiments in vitro/vivo.After subcutaneous injection with SART3 gene vaccine,cytotoxic T lymphocyte(CTL)activity in vitro was measured using Cell Counting Kit-8.As for the in vivo part,C3H mice were divided into several groups.One group was challenged with tumor cells after immunity.Another group was treated with the vaccine after tumor implantation.It was found that human SART3 DNA vaccine can elicit a specific CTL reaction from the mouse splenocytes.After vaccination,tumor occurrence and tumor growth speed was reduced.The vaccine also shows activity in tumor treatment.We con-clude that the human SART3 DNA vaccine can induce antitumor ability against tumor cells expressing human SART3(LM8-SART3)in vitro/vivo which may provide new possibilities in antitumor therapy.

Human bocavirus 1 and 2 genotype-specific antibodies for rapid antigen testing in pediatric patients with acute respiratory infections

Background Previous serological studies of human bocavirus(HBoV)1 could not exclude cross-reactivity with the other three HBoVs,particularly HBoV2.Methods To search for genotype-specific antibodies against HBoV1 and HBoV2,the divergent regions(DRs)located on the major capsid protein VP3 were defined through viral amino acid alignment and structure prediction.DR-deduced peptides were used as antigens to harvest corresponding anti-DR rabbit sera.To determine their genotype specificities for HBoV1 and HBoV2,these sera samples were used as antibodies against the antigens VP3 of HBoV1 and HBoV2(expressed in Escherichia coli)in western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and bio-layer interferometry(BLI)assays.Subsequently,the antibodies were evaluated with clinical specimens from pediatric patients with acute respiratory tract infection by indirect immunofluorescence assay(IFA).Results There were four DRs(DR1–4)located on VP3 with different secondary and tertiary structures between HBoV1 and HBoV2.Regarding the reactivity with VP3 of HBoV1 or HBoV2 in WB and ELISA,high intra-genotype cross-reactivity of anti-HBoV1 or HBoV2 DR1,DR3,and DR4,but not anti-DR2,was observed.Genotype-specific binding capacity of anti-DR2 sera was confirmed by BLI and IFA,in which only anti-HBoV1 DR2 antibody reacted with HBoV1-positive respiratory specimens.Conclusion Antibodies against DR2,located on VP3 of HBoV1 or HBoV2,were genotype specific for HBoV1 and HBoV2,respectively.

Effects of phosphatidylinositol 3-kinase inhibitor on humancervical carcinoma cells in vitro

Phosphatidylinositol 3-kinase(PI3K)is a crucial cell survival pathway implicated in tumorigenesis because of its role in stimulating cell proliferation and suppressing apoptosis.This study was to investigate the regulation of proliferation and apoptosis by LY294002,an inhibitor of PI3K in cervical cancer cells and the expression of FLICE-like inhibitory protein(c-FLIP)in vitro.Human cervical cancer HeLa cells were used in this experiment and cultured.The cultured cells were treated with LY294002 at different concentrations(10,25,50 and 100µmol/L)for 6,12,24,and 48 h before harvesting for evaluation.Cell viability was measured by 3-(4,5)-dimethylthiazol(-2-y1)-3,5-di-phenyltetrazoliumbromide(MTT)assay.Apoptosis was analyzed byflow cytometry.The expression of c-FLIP was detected by Western blot.Cell viability was inhibited by LY294002 significantly(P<0.05).Flow cytometry analysis revealed that cell apoptosis was significantly increased in the presence of LY294002 as compared with the control group.Although the expression of c-FLIP was increased in a short time,the expression of c-FLIP was markedly suppressed after the treatment of LY294002 for 48 h.These results suggested that the PI3K/Akt signal pathway might be involved in the regulation of cell apoptosis in cervical cancer cells.Moreover,the regulation of c-FLIP expression through PI3K/Akt signal pathway in cervical cancer cells was observed in vitro.

NRG1、HER3在前列腺癌组织中的表达及其与临床病理特征和预后的关系

目的研究前列腺癌(PC)组织中神经调节蛋白1(NRG1)、人表皮生长因子受体3(HER3)表达与临床病理特征及预后的关系。方法选取2015年2月—2020年2月吉林省人民医院泌尿外科诊治PC患者96例,免疫组织化学检测组织中NRG1、HER3表达;Kaplan-Meier曲线(Log-Rank检验)比较不同NRG1、HER3表达对PC患者预后的影响;COX回归分析PC患者预后的影响因素。结果PC癌组织中NRG1、HER3阳性率分别为78.13%(75/96)、75.00%(72/96),高于癌旁组织6.25%(6/96)、8.33%(8/96)(χ^(2)/P=101.670/<0.001,87.771/<0.001)。TNM分期Ⅲ期、Gleason评分>7分及术前PSA水平≥20μg/L患者癌组织中NRG1、HER3阳性率大于TNM分期Ⅰ~Ⅱ期、Gleason评分≤7分及术前PSA水平<20μg/L(χ^(2)/P=6.181/0.013,8.533/0.003;7.731/0.005,6.769/0.009;6.508/0.011,7.376/0.007)。NRG1阳性组、HER3阳性组3年累积无进展生存率分别低于NRG1阴性组、HER3阴性组(χ^(2)/P=4.267/0.039,5.499/0.019)。TNM分期Ⅲ期、Gleason评分>7分、术前PSA≥20μg/L、NRG1阳性,HER3阳性是影响PC患者预后的独立危险因素[OR(95%CI)=1.448(1.118~1.875),1.401(1.138~1.724),1.353(1.059~1.728),1.338(1.057~1.692),1.293(1.014~1.649)]。结论PC癌组织中NRG1、HER3表达升高,与PC不良临床病理特征相关,是新的评估PC预后的肿瘤标志物。

血清人神经趋化蛋白及人甲壳质酶蛋白40水平与老年阿尔茨海默症患者早期认知功能损害的关系

目的探讨血清人神经趋化蛋白(CX3CL1)、人甲壳质酶蛋白40(YKL-40)水平与老年阿尔茨海默症(AD)患者早期认知功能损害的关系。方法选取2021年2月—2023年12月新乡医学院第二附属医院收治的110例AD患者作为AD组,另选取本院同期健康体检者50例作为对照组。比较2组临床资料及血清CX3CL1、YKL-40水平,采用多因素Logistic回归模型分析AD患者认知功能损害的影响因素。根据简易精神状态量表(MMSE)评估结果将110例AD患者分为轻度认知障碍组(n=47)、中度认知障碍组(n=36)、重度认知障碍组(n=27),并采用Spearman相关性分析法分析血清CX3CL1、YKL-40与MMSE评分、Administration认知评估量表第3版(ACE-Ⅲ)评分、蒙特利尔认知评估量表(MoCA)评分的关系。结果AD组患者80岁以上比率、文化程度为小学及以下比率、吸烟史比率、饮酒史比率、合并糖尿病比率、合并高血压比率、AD家族史比率、独居比率及血清CX3CL1、YKL-40水平高于对照组,从不体育锻炼/体力劳动比率、MMSE评分、ACE-Ⅲ评分及MoCA评分低于对照组,差异有统计学意义(P<0.05)。高龄、合并糖尿病、血清CX3CL1、YKL-40是AD患者认知功能损害的独立危险因素(P<0.05),大专及以上文化程度为AD患者认知功能损害的保护因素(P<0.05)。与轻度认知障碍组相比,中度认知障碍组、重度认知障碍组血清CX3CL1、YKL-40水平偏高,与中度认知障碍组相比,重度认知障碍组血清CX3CL1、YKL-40水平偏高,差异有统计学意义(P<0.05)。Spearman相关性分析发现,血清CX3CL1、YKL-40与MMSE评分、ACE-Ⅲ评分、MoCA评分均呈负相关(P<0.05)。结论血清CX3CL1、YKL-40在老年AD患者体内呈高表达,且与老年AD患者早期认知功能损害关系密切。

环状RNA同源性蛋白激酶3靶向微RNA-338促进胶质瘤细胞侵袭、迁移的实验研究

目的探讨人血清环状RNA同源性蛋白激酶3(CircHIPK3)靶向微RNA-338(miR-338)对胶质瘤细胞U251细胞侵袭、迁移的影响。方法2021年2-12月,在武汉大学人民医院科研中心将U251细胞分为空白(NG)组、CircHIPK3阴性对照(shcontrol)组、HIPK3敲减(sh-CircHIPK3)组,实时荧光定量PCR(qRT-PCR)检测U251细胞中CircHIPK3、miR-338表达水平;Transwell检测细胞迁移与侵袭;划痕法检测细胞迁移;流式细胞术检测细胞周期;通过Circular RNA Interactome、RegRNA2.0、CircBank Database网站预测CircHIPK3(ID:hsa_circ_0000284)的靶向miRNA并用双萤光素酶实验验证,蛋白质印迹法检测基质金属蛋白酶(MMP)-2、MMP-9蛋白表达。结果与NG组、sh-control组比较,sh-CircHIPK3组中CircHIPK3(1.00±0.00、1.06±0.26比0.56±0.06)表达水平显著降低(P<0.05),miR-338(1.00±0.00、1.12±0.19比1.89±0.28)表达、G1期细胞比例[(58.72±0.36)%、(58.45±0.27)%比(64.72±0.47)%]升高(P<0.05),U251细胞侵袭数目[(164.89±12.55)个、(165.77±12.16)个比(80.13±11.37)个]、划痕愈合率[(25.66±2.37)%、(26.38±2.53)%比(10.36±1.53)%]、迁移细胞数目[(196.72±18.75)个、(194.65±17.86)个比(95.58±8.66)个]、S期细胞比例[(26.45±0.39)%、(26.57±0.41)%比(20.72±0.18)%]明显降低(P<0.05);miR-338是CircHIPK3的靶基因。与NG组、sh-control组比较,sh-CircHIPK3组MMP-2(1.31±0.23、1.33±0.20比0.61±0.05)、MMP-9(1.16±0.22、1.15±0.21比0.85±0.19)蛋白表达水平均显著降低(P<0.05)。结论沉默CircHIPK3通过靶向上调miR-338表达能抑制胶质瘤细胞U251细胞迁移和侵袭。

GATA3介导miR-21/PTEN轴对子宫内膜癌细胞增殖、侵袭的影响

目的分析GATA结合蛋白3(GATA3)介导微小RNA-21(miR-21)/人类第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)轴对子宫内膜癌细胞增殖、侵袭的影响。方法取HEC-1-A细胞,进行转染分组,分为对照组、GATA3空载质粒组、GATA3过表达质粒组、GATA3 siRNA阴性对照组、GATA3 siRNA组。检测各组细胞中GATA3、miR-21、PTEN表达量、增殖情况、凋亡率、迁移、侵袭。结果与hEEC组相比,HEC-1-A组、HEC-1-B组、Ishikawa组细胞中GATA3、miR-21表达水平升高,PTEN表达水平降低(P<0.05)。与GATA3空载质粒组相比,GATA3过表达质粒组GATA3、miR-21 mRNA表达量、增殖率、迁移距离、侵袭细胞数、Vimentin水平升高,PTEN mRNA表达量、凋亡率、Caspase-9、Bax、E-cadherin水平降低(P<0.05);与GATA3 siRNA阴性对照组相比,GATA3、miR-21 mRNA表达量、增殖率、迁移距离、侵袭细胞数、Vimentin水平降低,PTEN mRNA表达量、凋亡率、Caspase-9、Bax、E-cadherin水平升高(P<0.05)。结论下调GATA3表达,可对miR-21/PTEN轴进行调节,使HEC-1-A细胞的增殖减慢,促进HEC-1-A细胞的凋亡。

羧甲司坦口服溶液联合重组人干扰素α1b治疗小儿急性喘息性支气管炎的效果

目的分析急性喘息性支气管炎患儿接受重组人干扰素α1b单药与联合羧甲司坦口服溶液治疗的效果。方法回顾性分析2021年6月至2023年6月医院收治的100例急性喘息性支气管炎患儿资料,按不同治疗方案分为对照组、观察组,各50例。对照组接受重组人干扰素α1b治疗,观察组接受羧甲司坦口服溶液联合重组人干扰素α1b治疗。比较两组临床疗效、主要症状缓解时间、气道炎症相关因子[趋化因子配体3(CCL3)、高迁移率族蛋白B1(HMGB1)、α1-酸性糖蛋白(α1-AG)]、T淋巴细胞(CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+))及不良反应。结果观察组临床总有效率高于对照组(P<0.05)。治疗后观察组气促、喘息、咳嗽、肺部音等症状缓解时间降低(P<0.05)。治疗4、7 d后,两组CCL3、HMGB1、α1-AG较治疗前下降,且观察组低于对照组(P<0.05);两组CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)较治疗前升高,且观察组高于对照组(P<0.05)。两组不良反应发生率比较,差异无统计学意义(P>0.05)。结论羧甲司坦口服溶液联合重组人干扰素α1b治疗急性喘息性支气管炎,可抑制气道炎症,调节机体免疫,促进症状缓解,疗效确切,且安全性高。

穿心莲内酯靶向Akt信号干预HIV感染T细胞的效率研究

目的探讨穿心莲内酯靶向Akt信号干预人类免疫缺陷病毒(HIV)感染T细胞的效率。方法培养Jurkat T细胞,用WST-1试剂检测穿心莲内酯干预对Jurkat T细胞活性的影响。将Jurkat T细胞分为空白对照组、穿心莲内酯组、LY294002组(PI3K抑制剂)及穿心莲内酯联合LY294002组。各组药物分别干预24 h后,采用实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞PI3K、Akt mRNA相对表达量,采用Western blotting检测各组细胞p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量。制备HIV假病毒,用假病毒感染各组细胞,萤光素酶系统检测假病毒感染Jurkat T细胞的效率。结果WST-1试剂检测结果显示,随着穿心莲内酯浓度增加,Jurkat T细胞活力降低(P<0.05)。qRT-PCR和Western blotting检测结果显示,穿心莲内酯降低Jurkat T细胞PI3K及其下游Akt mRNA相对表达量及p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量(P<0.05)。HIV假病毒感染实验结果显示,穿心莲内酯降低HIV假病毒感染Jurkat T细胞的效率,联合组比单独组的抑制效果更好(P<0.05)。结论穿心莲内酯可能通过PI3K降低其下游Akt的活化,从而减少HIV假病毒在Jurkat T细胞间的传播及进入细胞的机会,降低其感染Jurkat T细胞的概率。

补骨脂素通过miR-101/PI3K/Akt轴对骨肉瘤细胞侵袭、转移的影响

目的探讨补骨脂素对骨肉瘤细胞侵袭、转移的抑制作用及可能的作用机制。方法用不同浓度补骨脂素作用于人骨肉瘤细胞(human osteosarcoma cells,MG-63),用CCK-8法检测细胞的存活情况,筛选最佳抑制浓度;用实时荧光定量法检测骨肉瘤组织与正常组织中微小RNA(microRNA,miR)-101的相对表达量。将对数生长期骨肉瘤细胞MG-63随机分为对照组、miR101模拟物阴性对照组(miR-NC组)、miR-101组、补骨脂素组和补骨脂素联合miR-101组。用四甲基偶氮唑盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT]实验检测细胞增殖抑制率;用肿瘤细胞侵袭实验(Transwell)检测细胞侵袭和迁移能力;用实时荧光定量聚合酶链式反应(quantitative real time polymerase chain reaction,RT-PCR)检测肌磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)mRNA的表达情况;用蛋白印迹法(Western blotting)检测磷酸化肌磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)的表达情况。结果与对照组比较,miR-101组、补骨脂素组和补骨脂素联合miR-101组的细胞增殖抑制率升高,细胞侵袭数量、细胞迁移数量、PI3K和Akt mRNA,p-PI3K、p-Akt、MMP2和MMP9蛋白的表达水平均降低(P<0.05)。与miR-101组和补骨脂素组比较,补骨脂素联合miR-101组的细胞增殖抑制率升高,细胞侵袭数量、细胞迁移数量、PI3K和Akt mRNA,p-PI3K、p-Akt、MMP-2和MMP-9蛋白的表达水平均降低(P<0.05)。结论补骨脂素能够降低骨肉瘤细胞MG-63的增殖能力,并抑制其侵袭和迁移能力,其作用机制可能与调节miR-101水平、影响PI3K/Akt信号通路有关。