AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.

Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

Regulatory potential of soil available carbon,nitrogen,and functional genes on N_(2)O emissions in two upland plantation systems

Dynamic nitrification and denitrification processes are affected by changes in soil redox conditions,and they play a vital role in regulating soil N_(2)O emissions in rice-based cultivation.It is imperative to understand the influences of different upland crop planting systems on soil N_(2)O emissions.In this study,we focused on two representative rotation systems in Central China:rapeseed–rice(RR)and wheat–rice(WR).We examined the biotic and abiotic processes underlying the impacts of these upland plantings on soil N_(2)O emissions.The results revealed that during the rapeseed-cultivated seasons in the RR rotation system,the average N_(2)O emissions were 1.24±0.20 and 0.81±0.11 kg N ha^(–1)for the first and second seasons,respectively.These values were comparable to the N_(2)O emissions observed during the first and second wheat-cultivated seasons in the WR rotation system(0.98±0.25 and 0.70±0.04 kg N ha^(–1),respectively).This suggests that upland cultivation has minimal impacts on soil N_(2)O emissions in the two rotation systems.Strong positive correlations were found between N_(2)O fluxes and soil ammonium(NH_(4)^(+)),nitrate(NO_(3)^(–)),microbial biomass nitrogen(MBN),and the ratio of soil dissolved organic carbon(DOC)to NO_(3)^(–)in both RR and WR rotation systems.Moreover,the presence of the AOA-amoA and nirK genes were positively associated with soil N_(2)O fluxes in the RR and WR systems,respectively.This implies that these genes may have different potential roles in facilitating microbial N_(2)O production in various upland plantation models.By using a structural equation model,we found that soil moisture,mineral N,MBN,and the AOA-amoA gene accounted for over 50%of the effects on N_(2)O emissions in the RR rotation system.In the WR rotation system,soil moisture,mineral N,MBN,and the AOA-amoA and nirK genes had a combined impact of over 70%on N_(2)O emissions.These findings demonstrate the interactive effects of functional genes and soil factors,including soil physical characteristics,available carbon and nitrogen,and their ratio,on soil N_(2)O emissions during upland cultivation seasons under rice-upland rotations.

应用Minigene剪接变异体分析技术诊断PMM2基因非经典剪接位点新变异的致病性

目的:研究Minigene剪接变异体分析技术在诊断磷酸甘露糖变位酶2(PMM2)相关先天性糖基化障碍(PMM2-CDG)中的价值,探讨磷酸甘露糖变位酶2(PMM2)基因剪接位点新变异对其转录产物的影响。方法:通过对1例PMM2-CDG患儿进行高通量测序查找可能的遗传学病因,利用Minigene剪接变异体分析技术,研究PMM2基因新剪接位点变异的致病性。根据美国医学遗传学与基因组学学会(ACMG)指南,判断新变异的致病性。结果:遗传学分析发现患儿系PMM2基因母源性c.691G>A(p.Val231Met)变异和父源性c.447+5G>A变异复合杂合子。Minigene剪接变异体分析发现:变异c.447+5G>A导致PMM2基因转录产物形成r.348_447del转录本,为致病性PMM2基因变异。患儿的临床特征为皮肤巩膜黄染,血清总胆红素、非结合胆红素和总胆汁酸明显升高,白蛋白明显降低,甲胎蛋白、铁蛋白和促甲状腺素等升高,对症支持治疗效果欠佳。结论:Minigene剪接变异体分析可为PMM2-CDG确诊和家系遗传咨询提供新的分子标记物,扩展了PMM2基因变异谱,为该病的临床诊治提供新的参考依据。

Vanillylacetone attenuates cadmium chloride-induced hippocampal damage and memory loss through upregulation of nuclear factor erythroid 2-related factor 2 gene and protein expression

Memory loss and dementia are major public health concerns with a substantial economic burden.Oxidative stress has been shown to play a crucial role in the pathophysiology of hippocampal damage-induced memory impairment.To investigate whether the antioxidant and anti-inflammatory compound vanillyla cetone(zingerone) can protect against hippocampal damage and memory loss induced by cadmium chloride(CdCl_(2)) administration in rats,we explo red the potential involvement of the nuclear factor erythroid 2-related factor 2(Nrf2) signaling pathway,which is known to modulate oxidative stress and inflammation.Sixty healt hy male Wistar rats were divided into five groups:vehicle-treated(control),vanillylacetone,CdCl_(2),vanillylacetone+ CdCl_(2),vanillylacetone+ CdCl_(2)+ brusatol(a selective pharmacological N rf2inhibitor) groups.Vanillylacetone effectively attenuated CdCl_(2)-induced damage in the dental gyrus of the hippocampus and improved the memory function assessed by the Morris Water Maze test.Additionally,vanillylacetone markedly decreased the hippocampal tissue levels of inflammatory biomarkers(interleukin-6,tumor necrosis factor-α,intracellular cell adhesive molecules) and apoptosis biomarkers(Bax and cleaved caspase-3).The control and CdCl_(2)-treated groups treated with va nillylacetone showed reduced generation of reactive oxygen species,decreased malondialdehyde levels,and increased superoxide dismutase and glutathione activities,along with significant elevation of nuclear Nrf2 mRNA and protein expression in hippocampal tissue.All the protective effects of vanillylacetone we re substantially blocked by the co-administration of brusatol(a selective N rf2 inhibitor).Va nillylacetone mitigated hippocampal damage and memory loss induced by CdCl_(2),at least in part, by activating the nuclear transcription factor Nrf2.Additionally,vanillylacetone exerted its potent antioxidant and antiinflammatory actions.

To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Pathogenesis of chronic enteropathy associated with the SLCO2A1 gene:Hypotheses and conundrums

Chronic enteropathy associated with the SLCO2A1 gene(CEAS)is a complex gastroenterological condition characterized by multiple ulcers in the small intestine with chronic bleeding and protein loss.This review explores the potential mechanisms underlying the pathogenesis of CEAS,focusing on the role of SLCO2A1-encoded prostaglandin transporter OATP2A1 and its impact on prostaglandin E2(PGE2)levels.Studies have suggested that elevated PGE2 levels contribute to mucosal damage,inflammation,and disruption of the intestinal barrier.The effects of PGE2 on macrophage activation and Maxi-Cl channel functionality,as well as its interaction with nonsteroidal anti-inflammatory drugs play crucial roles in the progression of CEAS.Understanding the balance between its protective and pro-inflammatory effects and the complex interactions within the gastrointestinal tract can shed light on potential therapeutic targets for CEAS and guide the development of novel,targeted therapies.

Transglutaminase 2 serves as a pathogenic hub gene of KRAS mutant colon cancer based on integrated analysis

BACKGROUND Colon cancer is acknowledged as one of the most common malignancies worldwide,ranking third in United States regarding incidence and mortality.Notably,approximately 40%of colon cancer cases harbor oncogenic KRAS mutations,resulting in the continuous activation of epidermal growth factor receptor signaling.AIM To investigate the key pathogenic genes in KRAS mutant colon cancer holds considerable importance.METHODS Weighted gene co-expression network analysis,in combination with additional bioinformatics analysis,were conducted to screen the key factors driving the progression of KRAS mutant colon cancer.Meanwhile,various in vitro experiments were also conducted to explore the biological function of transglutaminase 2(TGM2).RESULTS Integrated analysis demonstrated that TGM2 acted as an independent prognostic factor for progression-free survival.Immunohistochemical analysis on tissue microarrays revealed that TGM2 was associated with an elevated probability of perineural invasion in patients with KRAS mutant colon cancer.Additionally,biological roles of the key gene TGM2 was also assessed,suggesting that the downregulation of TGM2 attenuated the proliferation,invasion,and migration of the KRAS mutant colon cancer cell line.CONCLUSION This study underscores the potential significance of TGM2 in the progression of KRAS mutant colon cancer.This insight not only offers a theoretical foundation for therapeutic approaches but also highlights the need for additional clinical trials and fundamental research to support our preliminary findings.

Gene expression analysis of cytokines and MMPs in melatonin and rhBMP-2 enhanced bone remodeling

BACKGROUND In the medical and dental fields,there is a need for studies of new therapeutic approaches for the treatment of bone defects that cause extensive bone loss.Melatonin may be an important endogenous biological factor for bone remodeling,and growth factors may enhance the repair process.AIM To evaluate the gene expression of cytokines(IL-1β,IL-6,IL-10 and TNF-α),markers of osteoclastogenesis(RANK,RANKL and OPG)and MMPs(MMP-1,MMP-2,MMP-8 and MMP-13)from the treatment of melatonin associated with an osteogenic membrane and rhBMP-2 on the recovery of a bone injury.METHODS Sixty-four rats were used and divided into 9 experimental groups and were formed according to the treatment carried out in the region of the bone lesion,which varied between the combination of 1,10 and 100μmol/L of melatonin.Gene Expression analysis was performed using real time-PCR by reading the concentration of total RNA and reverse transcription.RESULTS There were differences between groups when compared with clot or scaffold control,and improvement with a higher concentration of melatonin or rhBMP-2.The combination melatonin(1μg)with 5μg of rhBMP-2,using the guided bone regeneration technique,demonstrated some effects,albeit mild,on bone repair of critical bone defects.CONCLUSION This indicates that the approach for administering these substances needs to be reassessed,with the goal of ensuring their direct application to the affected area.Therefore,future research must be carried out,seeking to produce materials with these ideal characteristics.

Identification of M2 macrophage-related genes for establishing a prognostic model in pancreatic cancer: FCGR3A as key gene

Background:Pancreatic ductal adenocarcinoma(PDAC)has a rich and complex tumor immune microenvironment(TIME).M2 macrophages are among the most extensively infiltrated immune cells in the TIME and are necessary for the growth and migration of cancers.However,the mechanisms and targets mediating M2 macrophage infiltration in pancreatic cancer remain elusive.Methods:The M2 macrophage infiltration score of patients was assessed using the xCell algorithm.Using weighted gene co-expression network analysis(WGCNA),module genes associated with M2 macrophages were identified,and a predictive model was designed.The variations in immunological cell patterns,cancer mutations,and enrichment pathways between the cohorts with the high-and low-risk were examined.Additionally,the expression of FCGR3A and RNASE2,as well as their association with M2 macrophages were evaluated using the HPA,TNMplot,and GEPIA2 databases and verified by tissue immunofluorescence staining.Moreover,in vitro cell experiments were conducted,where FCGR3A was knocked down in pancreatic cancer cells using siRNA to analyze its effects on M2 macrophage infiltration,tumor proliferation,and metastasis.Results:The prognosis of patients in high-risk and low-risk groups was successfully distinguished using a prognostic risk score model of M2 macrophage-related genes(p=0.024).Between the high-and low-risk cohorts,there have been notable variations in immune cell infiltration patterns,tumor mutations,and biological functions.The risk score was linked to the manifestation of prevalent immunological checkpoints,immunological scores,and stroma values(all p<0.05).In vitro experiments and tissue immunofluorescence staining revealed that FCGR3A can promote the infiltration or polarization of M2 macrophages and enhance tumor proliferation and migration.Conclusions:In this study,an M2 macrophage-related pancreatic cancer risk score model was established,and found that FCGR3A was correlated with tumor formation,metastasis,and M2 macrophage infiltration.

PRRT2基因突变相关疾病谱的临床特征及遗传学分析

目的回顾性分析总结10例PRRT2基因突变相关疾病谱患者的临床特点及遗传学特征。方法收集2020-07—2022-08就诊于首都医科大学宣武医院儿科的10例PRRT2基因相关癫痫患儿的临床特征、脑电图、头颅磁共振检查、基因特征及治疗结果,回顾性分析其特征。结果10例患儿均提示PRRT2基因杂合突变,其中3例为片段缺失,5例为移码突变,1例为剪接突变。10例患者中男5例,女5例,起病年龄4个月~10岁,其中7例诊断为自限性家族性婴儿癫痫(SFIE),1例诊断为发作性运动诱发性运动障碍(PKD),2例诊断为伴婴儿惊厥的发作性运动诱发性运动障碍(IC/PKD);5例存在PRRT2基因突变相关疾病家族史。7例口服奥卡西平治疗后发作控制,3例口服左乙拉西坦治疗后发作控制。结论PKD、SFIE及IC/PKD是一组与PRRT2基因突变相关的疾病谱,c.649dupC基因突变位点是热点突变位点。对于高度考虑SFIE、PKD及IC/PKD的患者,如全外显子测序未发现异常基因,需进一步行内含子及染色体微缺失/微重复检测,达到早期诊断及治疗的目的。

人子宫颈癌基因HCCR-2反义真核表达载体的构建及鉴定

目的克隆人子宫癌基因HCCR-2并构建其反义真核表达载体。方法从人肝癌细胞株HepG2提取RNA,利用RT-PCR方法,克隆出一包括HCCR-2编码区的长约1kb的片段,并将其与T载体连接,测序证实无误后,用SalⅠ、SacⅡ双酶切重组T载体,然后将该片段反向插入真核表达载体pIRES2-EGFP的多克隆位点,最后对重组真核表达载体进行双酶切鉴定。结果RT-PCR扩增出一长约1kb的HCCR-2片段,琼脂糖凝胶电泳显示目的片段成功反向连接到pIRES2-EGFP载体上。结论成功克隆了HCCR-2基因并构建了其反义真核表达载体pIRES2-EGFP/HCCR-2(-)。

HCCR-2干扰真核表达载体的构建及其稳定转染细胞系的建立

目的构建HCCR-2干扰真核表达载体,获得干扰质粒稳定转染的HepG2细胞系。方法人工合成HCCR-2基因干扰序列并定向插入到RNAi真核表达载体pGenesil-1,通过测序鉴定。将干扰质粒用脂质体法转染HepG2细胞,经G418持续压力选择和有限稀释法获得稳定转染的细胞系。RT-PCR检测筛选克隆HCCR-2 mRNA的表达水平。结果测序表明HCCR-2干扰序列及读框完全正确,干扰质粒稳定转染的HepG2细胞系在倒置荧光显微镜下呈绿色荧光。RT-PCR结果显示RNAi-H1、RNAi-H3序列对HCCR-2 mRNA有较好的抑制效果。结论成功构建了HCCR-2干扰真核表达载体,HCCR-2干扰质粒稳定转染的HepG2细胞系的建立为进一步研究HCCR-2在肝癌细胞中的作用奠定了基础。

人子宫颈癌基因HCCR-2真核表达载体的构建及鉴定

目的:克隆人子宫癌基因HCCR-2并构建其真核表达载体,为研究其在人胃癌细胞中与P53的相互作用关系奠定基础.方法:从人肝癌细胞株HepG2提取RNA,利用RT-PCR方法,先克隆出一包括HCCR-2编码区的长约1kb的片段,并将其与T载体连接并转化,测序证实无误后,以HCCR-2/T载体为模板,再通过PCR克隆出HCCR-2的编码区序列,将其连接到pIRES2-EGFP真核表达载体,并通过测序获得证实.结果:RT-PCR扩增出一长约1kb的HCCR-2片段,PCR扩增出约0.9kb大小的目的片段,HCCR-2/T,pIRES2-EGFP/HCCR-2(+)DNA测序均表明编码序列及读框完全正确.结论:成功构建HCCR-2真核表达载体pIRES2-EGFP/HCCR-2(+),为进一步研究其致癌机制打下基础.

Main Agronomic Characters and Grain Quality of Rice Blast Resistance Gene Pi-d2 Transgenic Rice

[Objective] The aim of this study was to provide metabolic evidence for the analysis of the ecological and safety assessment of Pi-d2-transgenic rice.[Method] The main agronomic characters of Pi-d2-transgenic rice were observed in field experiment and the grain chemical characters and amino acid content were measured.[Results] Introduction of foreign gene Pi-d2 resulted in stably hereditable variation in agronomic characteristics in the descents.Most of the transgenic lines grew normally and orderly.Compared with the control（wild type plants）,about half of transgenic plants showed an increased or reduced plant height.There was no observable difference between transgenic plants and controls in tiller number,length of panicle,panicles per plant,seed-setting rate and 1 000-grain weight.Total amino acid content in transgenic rice was reduced,while the starch content,GC and GT were not altered in comparison with the control.[Conclusion] Introduction of foreign gene Pi-d2 has remarkable influence on plant height,while little on grain chemical characters.

Effects of Light and Temperature on the Expression of the Lhcb2 Gene in Pea

An approximately 800 bp cDNA ( Lhcb 2) encoding light_harvesting chlorophyll a/b_binding protein complex (type Ⅱ) was cloned from the seedling of pea ( Pisum sativum L.) with RT_PCR method. Southern blotting using special probe demonstrated that there existed one copy of Lhcb 2 in pea genome. RT_PCR and Northern blotting revealed the expression of Lhcb 2 which was regulated by light in a time_dependent expression manner. The Lhcb 2 gene didn't express untill 2 h after irradiated with white light. Low temperature (4 ℃) also affected the Lhcb 2 gene by decreasing half of its expression under 25 ℃.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

TGF-β2和geneX对BrdU标记骨髓间充质干细胞增殖与成骨分化的作用

目的研究TGF-β2和gene X对Brd U标记的骨髓间充质干细胞增殖与成骨分化作用的影响.方法采用全骨髓贴壁培养法分离骨髓间充质干细胞,传代并鉴定,设实验组和空白对照组,实验组分4组:A组:Brd U标记后的BMSCs组;B组:Brd U标记后的BMSCs+TGF-β2组;C组:Brd U标记后的BMSCs+gene X组;D组:Brd U标记后的BMSCs+gene X+TGF-β2组.空白对照组为Brd U标记后的单纯BMSCs加入基础培养基避光培养.诱导培养后观察骨髓间充质干细胞生长形态、检测生长动力学(methyl thiazolyl tetrazolium,MTT)、碱性磷酸酶(ALP)和钙定量、进行细胞Von-Kossa法染色,观察成骨分化作用.结果 (1)4组BMSCs吸光度值(OD)分析比较显示:细胞组间差异有统计学意义(P<0.05);(2)各组BMSCs成骨诱导后碱性磷酸酶(ALP)和钙定量检测结果显示:细胞组间差异有统计学意义(P<0.05);(3)进行细胞Von-Kossa法染色,D组可见大量钙结节;C组可见较多钙结节;B组可见部分钙结节;A组可见少量钙结节;空白对照组未见钙结节.结论 (1)TGF-β2可促进骨髓间充质干细胞的增殖和诱导其向成骨细胞分化;(2)gene X可促进骨髓间充质干细胞向成骨细胞分化,且在细胞因子TGF-β2联合作用下成骨分化作用更明显.

Study on the Cloning and Isolation of sus scrofa GPX2 Gene by RACE Method

[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was D098982）.

反义HCCR-2转染对肝癌细胞HepG2形态的影响

目的研究HCCR-2反义基因转染对肝癌HepG2细胞株形态学的影响。方法用脂质体法将HCCR-2反义真核表达载体导入HepG2细胞,从光镜、HE染色、荧光显微镜、电镜水平观察转染前后细胞形态学的改变。结果与亲本细胞相比,光镜及HE染色下转染HCCR-2反义基因的HepG2细胞变得狭长,胞体增大,胞浆丰富,分裂相减少,核浆比下降,异型性减少;荧光显微镜下反义基因转染细胞发出绿色荧光,透射电镜下反义基因转染细胞出现凋亡。结论 HCCR-2反义基因可以促进HepG2细胞的分化。