Müller cells are activated in response to retinal outer nuclear layer degeneration in rats subjected to simulated weightlessness conditions

A microgravity environment has been shown to cause ocular damage and affect visual acuity,but the underlying mechanisms remain unclear.Therefore,we established an animal model of weightlessness via tail suspension to examine the pathological changes and molecular mechanisms of retinal damage under microgravity.After 4 weeks of tail suspension,there were no notable alterations in retinal function and morphology,while after 8 weeks of tail suspension,significant reductions in retinal function were observed,and the outer nuclear layer was thinner,with abundant apoptotic cells.To investigate the mechanism underlying the degenerative changes that occurred in the outer nuclear layer of the retina,proteomics was used to analyze differentially expressed proteins in rat retinas after 8 weeks of tail suspension.The results showed that the expression levels of fibroblast growth factor 2(also known as basic fibroblast growth factor)and glial fibrillary acidic protein,which are closely related to Müller cell activation,were significantly upregulated.In addition,Müller cell regeneration and Müller cell gliosis were observed after 4 and 8 weeks,respectively,of simulated weightlessness.These findings indicate that Müller cells play an important regulatory role in retinal outer nuclear layer degeneration during weightlessness.

Overexpressing NeuroD1 reprograms Müller cells into various types of retinal neurons

The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7 m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose-and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.

Vaginal clear cell adenocarcinoma in Herlyn-Werner-Wunderlich syndrome:A case report

BACKGROUND Herlyn-Werner-Wunderlich(HWW)syndrome is a rare Müllerian duct anomaly,characterized by a combination of urogenital abnormalities.The occurrence of primary cervico-vaginal carcinomas in patients with HWW syndrome is excep-tionally rare,posing significant challenges for screening,early diagnosis,and effective management.CASE SUMMARY We report a rare case of primary clear cell carcinoma of the vagina complicated in a 40-year-old woman with HWW syndrome.The patient presented with irregular vaginal bleeding for 4 years.On gynecological examination,an oblique vaginal septum was suspected.Surgical resection of the vaginal septum revealed a com-municating fistula and a tumor on the left vagina and the left side of the septum,which was confirmed as clear cell carcinoma.One month later,she underwent a radical hysterectomy,vaginectomy,bilateral salpingo-oophorectomy,and pelvic lymph node dissection.Due to significant side effects,she completed only one course of chemotherapy.A year later,lung metastasis was detected and continued to grow.A thoracoscopic wedge resection of the right upper lobe was performed 4 years after the initial surgery.We also conducted a systemic review of the lite-rature on primary cervical or vaginal carcinoma in HWW syndrome to explore this rare entity.CONCLUSION Cervico-vaginal adenocarcinomas in patients with HWW syndrome are occult,and require early surgical intervention and regular imaging surveillance.

基于METTL3介导的miR-29a-3p的m^(6)A修饰探讨平喘颗粒抑制气道上皮细胞泛凋亡治疗哮喘的机制研究

目的:观察平喘颗粒对METTL3介导的气道上皮细胞中miR-29a-3p的m^(6)A修饰的影响,明确其抑制哮喘气道炎症的分子机制。方法:16HBE采用LPS诱导(50 mg/L)构建细胞模型,并给予平喘颗粒含药血清和地塞米松进行干预。分别采用CCK8法检测细胞活性、ELISA法检测炎症因子(TNF-α、IL-6和IL-8)的含量和miR-29a-3p的m^(6)A修饰水平、Western blot检测METTL3与泛凋亡蛋白的表达和qRT-PCR检测METTL3与miR-29a-3p的表达。结果:与模型组相比,平喘颗粒能够显著增加16HBE的活力,抑制炎症因子TNF-α、IL-6和IL-8的含量,下调泛凋亡相关蛋白p-RIPK3、p-MLKL、cleaved Caspase-1、cleaved Caspase-3的表达和GSDMD-NT/FL-GSDMD与GSDME-NT/FL-GSDME的比值,差异均具有统计学意义(P<0.01)。此外,平喘颗粒能够显著上调细胞中miR-29a-3p和METTL3的表达水平,促进miR-29a-3p的m^(6)A修饰水平,与模型组相比差异均具有统计学意义(P<0.01)。结论:平喘颗粒主要通过METTL3增强miR-29a-3p的m^(6)A修饰水平,抑制气道上皮细胞泛凋亡,改善气道炎症。

Cadmium Activates Reactive Oxygen Species-dependent AKT/mT OR and Mitochondrial Apoptotic Pathways in Neuronal Cells

Objective To examine the role of Cd-induced reactive oxygen species（ROS） generation in the apoptosis of neuronal cells. Methods Neuronal cells（primary rat cerebral cortical neurons and PC12 cells） were incubated with or without Cd post-pretreatment with rapamycin（Rap） or N-acetyl-L-cysteine（NAC）. Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B（Akt）/mammalian target of rapamycin（m TOR） and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase（p70 S6K）, and eukaryotic initiation factor 4E binding protein 1（4E-BP1）. Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein（Bcl-2/Bax） ratio, release of cytochrome c, cleavage of caspase-3 and poly（ADP-ribose） polymerase（PARP）, and nuclear translocation of apoptosis-inducing factor（AIF） and endonuclease G（Endo G）. Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

Intestinal M cells:The fallible sentinels?

The gastrointestinal tract represents the largest mucosal membrane surface in the human body. The immune system in the gut is the first line of host defense against mucosal microbial pathogens and it plays a crucial role in maintaining mucosal homeostasis. Membranous or microfold cells, commonly referred to as microfold cells, are specialized epithelial cells of the gut-associated lymphoid tissues (GALT) and they play a sentinel role for the intestinal immune system by delivering luminal antigens through the follicle-associated epithelium to the underlying immune cells. M cells sample and uptake antigens at their apical membrane, encase them in vesicles to transport them to the basolateral membrane of M cells, and from there deliver antigens to the nearby lymphocytes. On the flip side, some intestinal pathogens exploit M cells as their portal of entry to invade the host and cause infections. In this article, we briefly review our current knowledge on the morphology, development, and function of M cells, with an emphasis on their dual role in the pathogenesis of gut infection and in the development of host mucosal immunity.

Modulation of TGFβ_2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye

AIM: To investigate the effect of protein kinase C (PKC) on transforming growth factor-β2 (TGFβ2) and dopamine in retinal Müller cells of guinea pig myopic eye. METHODS: Myopia was induced by translucent goggles in guinea pig, whose retinal Müller cells were cultured using the enzyme-digesting method. Retinal Müller cells were divided into 5 groups: normal control, myopia, myopia plus GF109203X, myopia plus PMA, myopia plus DMSO. PKC activities were detected by the non-radioactive methods. TGFβ2 and tyrosine hydroxylase (TH) proteins were analyzed by Western Blotting in retinal Müller cells. Dopamine was determined by the high-performance liquid chromatography- electrochemical detection in suspensions. RESULTS: After 14 days deprived, the occluded eyes became myopic with ocular axle elongating. Müller cells of guinea pigs were obtained using enzyme digestion. Compared with normal control group, the increase in PKC activity and the up-regulation in TGFβ2 expression were found in retinal Müller cells of myopic eyes, with the decrease of TH and dopamine content (P <0.05). After PKC activated by PMA, TGFβ2 and TH content were up-regulated with the increase of dopamine content (P <0.05). While the PKC activities was inhibited by GF109203X, proteins of TGFβ2 and TH were down-regulated in the myopic eyes, with the decrease of dopamine content (P <0.05). CONCLUSION: TGFβ2 and dopamine are modulated by PKC in Müller cells of the myopic eyes in guinea pig.

Targeting lentiviral vectors to primordial germ cells(PGCs):An efficient strategy for generating transgenic chickens

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

Pirfenidone suppresses the abnormal activation of human Müller cells after platelet-derived growth factor-BB stimulation

AIM: To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB(PDGF-BB). METHODS: The primary human Müller cells were separated from retinal tissues and established the pathogenic model by stimulated with PDGF-BB. The Müller cells behaviour of normal group and the model group was measured by MTT assay, Trypan blue assay, cell migration assay, and collagen contraction assay. The expression of transforming growth factor(TGF)-β1,-β2, and pigment epithelium-derived factor(PEDF) was estimated with realtime polymerase chain reaction(PCR), Western blot and immunofluorescence analyses. RESULTS: A pathogenic/proliferative model of Müller cells was established by stimulating normal cultured Müller cells with 10 ng/mL PDGF-BB for 48 h. After treated with 0.2 and 0.3 mg/mL pirfenidone, the proliferation, migration and collagen contraction was statistically significantly depressed in the model group compared with the normal groups. The expression levels of TGF-β1 and TGF-β2 were significantly down-regulated, while the PEDF expression was significantly up-regulated after treated with 0.2 and 0.3 mg/mL pirfenidone in the model group. CONCLUSION: Pirfenidone effectively suppress the proliferation, migration and collagen contraction of the human Müller cells stimulated with PDGF-BB through down-regulation of TGF-β1/TGF-β2 and up-regulation of PEDF.

禽流感病毒M基因的重组慢病毒构建

旨在建立针对禽流感病毒(AIV)的实验室快速分子检测方法。构建重组慢病毒作为分子检测方法中的阳性对照品,采用PCR扩增技术获得AIV M基因的保守区片段,并构建pLVX-M-IRES-ZsGreen1重组慢病毒穿梭载体;将鉴定成功的重组穿梭质粒与骨架质粒pMD2.G和psPAX2共转染293T细胞,获得携带AIV M基因保守区的重组慢病毒;将重组慢病毒感染293T细胞,观察其荧光表达情况,收集、浓缩病毒液并测定其滴度;用AIV实时荧光定量PCR(RT-qPCR)检测方法检测M基因。结果表明:携带AIV M基因的重组慢病毒成功拯救,且其病毒滴度高、安全性强,可作为分子检测方法的阳性对照标准品。综上,本研究成功构建了AIV M基因的重组慢病毒,可高效包装出含有M基因保守区的慢病毒颗粒,为后续AIV分子检测试剂盒的研制提供参考。

Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells-possibilities and current limitations

BACKGROUND Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients.They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells(RGCs)and Müller glia.AIM To refine human-induced pluripotent stem cells(hiPSCs)differentiated into threedimensional(3D)retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses.METHODS In this study we described,evaluated,and refined methods with which to generate Müller glia and RGC progenitors,isolated them via magnetic-activated cell sorting,and assessed their lineage stability after prolonged 2D culture.Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry,and the ultrastructural composition of retinal organoid cells was investigated.RESULTS Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids.Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers.CONCLUSION Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Prion Protein Binds to Aldolase A Produced by Bovine Intestinal M Cells

Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.

Modulating poststroke inflammatory mechanisms: Novel aspects of mesenchymal stem cells, extracellular vesicles and microglia

Inflammation plays an important role in the pathological process of ischemic stroke,and systemic inflammation affects patient prognosis.As resident immune cells in the brain,microglia are significantly involved in immune defense and tissue repair under various pathological conditions,including cerebral ischemia.Although the differentiation of M1 and M2 microglia is certainly oversimplified,changing the activation state of microglia appears to be an intriguing therapeutic strategy for cerebral ischemia.Recent evidence indicates that both mesenchymal stem cells(MSCs)and MSC-derived extracellular vesicles(EVs)regulate inflammation and modify tissue repair under preclinical stroke conditions.However,the precise mechanisms of these signaling pathways,especially in the context of the mutual interaction between MSCs or MSC-derived EVs and resident microglia,have not been sufficiently unveiled.Hence,this review summarizes the state-ofthe-art knowledge on MSC-and MSC-EV-mediated regulation of microglial activity under ischemic stroke conditions with respect to various signaling pathways,including cytokines,neurotrophic factors,transcription factors,and microRNAs.

二烯丙基二硫通过磷酸化Chk1负调控Cdc25C/CyclinB1/CDK1通路阻滞白血病K562细胞G_(2)/M期

目的:研究二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病K562细胞周期阻滞及其分子机制。方法:采用CCK-8、细胞计数及流式细胞术观察DADS对K562细胞增殖与周期阻滞效应。Western Blot检测DADS对K562细胞PCNA、Chk1/2以及下游分子Cdc25C、CyclinB1与CDK1表达的影响。结果:CCK-8检测显示,15μmol/L、30μmol/L、60μmol/L、120μmol/L、240μmol/L DADS处理后,呈浓度依赖性抑制K562细胞增殖,抑制率分别为32.48%、59.34%、66.42%、77.06%、81.05%(P<0.05)。对照组与Tween80组无抑制作用(P>0.05)。细胞计数结果显示,30μmol/L、60μmol/L与120μmol/L DADS处理K562细胞后,群体倍增时间分别为22.71±0.29、36.69±0.93与73.02±0.87呈浓度依赖性增加(P<0.05),而Tween80组与对照组无明显差异(P>0.05)。流式细胞术检测显示,60μmol/L与120μmol/L DADS作用K562细胞24 h与48 h后,G_(2)/M期百分率分别增加到17.6%与28.5%和18.6%与34.4%,较对照组有显著性差异(P<0.05)。60μmol/L DADS作用K562细胞1 h、2 h、4 h、8 h和24 h后,PCNA表达呈时间依赖性表达下调(P<0.05)。p-Chk1表达呈时间依赖性上调(P<0.05),而Chk1、Chk2与p-Chk2表达无明显差异(P>0.05)。并且,Cdc25C、CyclinB1和CDK1分别呈时间依赖性下调(P<0.05),但是,14-3-3蛋白无明显改变(P>0.05)。结论:DADS可磷酸化Chk1通过Cdc25C/CyclinB1/CDK1通路抑制K562细胞增殖与阻滞G_(2)/M期。

Homer1a reduces inflammatory response after retinal ischemia/reperfusion injury

Elevated intraocular pressure(IOP)is one of the causes of retinal ischemia/reperfusion injury,which results in NRP3 inflammasome activation and leads to visual damage.Homerla is repo rted to play a protective role in neuroinflammation in the cerebrum.However,the effects of Homerla on NLRP3inflammasomes in retinal ischemia/reperfusion injury caused by elevated IOP remain unknown.In our study,animal models we re constructed using C57BL/6J and Homer1^(flox/-)/Homerla^(+/-)/Nestin-Cre^(+/-)mice with elevated IOP-induced retinal ischemia/repe rfusion injury.For in vitro expe riments,the oxygen-glucose deprivation/repe rfusion injury model was constructed with M uller cells.We found that Homerla ove rexpression amelio rated the decreases in retinal thickness and Muller cell viability after ischemia/reperfusion injury.Furthermore,Homerla knockdown promoted NF-κB P65^(Ser536)activation via caspase-8,NF-κB P65 nuclear translocation,NLRP3 inflammasome formation,and the production and processing of interleukin-1βand inte rleukin-18.The opposite results we re observed with Homerla ove rexpression.Finally,the combined administration of Homerla protein and JSH-23 significantly inhibited the reduction in retinal thickness in Homer1^(flox/-)Homer1a^(+/-)/Nestin-Cre^(+/-)mice and apoptosis in M uller cells after ischemia/reperfusion injury.Taken together,these studies demonstrate that Homer1a exerts protective effects on retinal tissue and M uller cells via the caspase-8/NF-KB P65/NLRP3 pathway after I/R injury.

Potential role of Müller cells in the pathogenesis of macropsia associated with epiretinal membrane:a hypothesis revisited

Pathophysiological explanations for metamorphopsia associated with retinal pathologies generally focus on photoreceptor organization disruption. However, the retinal microarchitecture is complicated, and we hypothesize that other retinal cells may also be involved. Metamorphopsia has been widely studied in eyes with epiretinal membranes and we revisit the idea that Müller cell displacement causes retinal macropsia. A Pub Med query and related article search for the macula ultrastructure under normal and pathological conditions revealed an enormous amount of information, particularly ultrahigh definition optical coherence tomography and other retinal imaging modality studies. Findings of these imaging studies support our hypothesis that Müller cells, and not cone photoreceptors, are primarily responsible for macropsia in eyes with epiretinal membranes. More specifically, we conclude that displacement of Müller cell endfeet, and not photoreceptor cones, is a more likely the explanation for retinal macropsia associated with epiretinal membranes.

The ROCK pathway inhibitor Y-27632 mitigates hypoxia and oxidative stress-induced injury to retinal Müller cells

Rho kinase （ROCK） was the first downstream Rho effector found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation. There is abundant evidence that the ROCK pathway participates in the pathogenesis of retinal endothelial injury and proliferative epiretinal membrane traction. In this study, we investigated the effect of the ROCK pathway inhibitor Y-27632 on retinal Müller cells subjected to hypoxia or oxidative stress. Müller cells were subjected to hypoxia or oxidative stress by exposure to CoCl2 or H2O2. After a 24-hour treatment with Y-27632, the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to assess the survival of Müller cells. Hoechst 33258 was used to detect apoptosis, while 2′,7′-dichlorodihydrofluorescein diacetate was used to measure reactive oxygen species generation. A transwell chamber system was used to examine the migration ability of Müller cells. Western blot assay was used to detect the expression levels of α-smooth muscle actin, glutamine synthetase and vimentin. After treatment with Y-27632, Müller cells subjected to hypoxia or oxidative stress exhibited a morphology similar to control cells. Y-27632 reduced apoptosis, α-smooth muscle actin expression and reactive oxygen species generation under oxidative stress, and it reduced cell migration under hypoxia. Y-27632 also upregulated glutamine synthetase expression under hypoxia but did not impact vimentin expression. These findings suggest that Y-27632 protects Müller cells against cellular injury caused by oxidative stress and hypoxia by inhibiting the ROCK pathway.

人参皂苷Rb1通过载脂蛋白M/线粒体凋亡途径对肝癌的影响及机制研究

目的:探究载脂蛋白M(ApoM)/线粒体凋亡在肝癌中的作用,以及人参皂苷Rb1(Rb1)通过ApoM/线粒体凋亡途径对肝癌影响的潜在机制。方法:生物信息学分析筛选ApoM在肝癌中的差异性表达及临床验证;分子对接技术确定Rb1与ApoM的靶向结合,体外培养人肝癌细胞(HepG2),并进行细胞活力检测(CCK-8)筛选Rb1最佳干预浓度,克隆实验用于检测HepG2细胞的增殖情况,划痕实验用于检测HepG2细胞的迁移能力,采用蛋白质印迹法(Western Blotting)检测ApoM及线粒体凋亡相关基因Bcl2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl2)、胱天蛋白酶-3(Caspase-3)、胱天蛋白酶-9(Caspase-9)蛋白表达,实时萤光定量聚合酶链式反应(RT-qPCR)法检测ApoM及线粒体凋亡相关基因Bax、Bcl2、Caspase-3、Caspase-9 mRNA表达水平。结果:生存分析发现ApoM低表达肝癌预后更差,分子对接提示Rb1与ApoM之间具有较高亲和力,克隆及划痕实验提示Rb1有效抑制HepG2细胞增殖,Western Blotting及RT-qPCR验证Rb1可以干预线粒体凋亡相关基因蛋白和mRNA表达。结论:揭示了ApoM/线粒体凋亡在肝癌中发挥重要作用,人参皂苷Rb1可能通过ApoM/线粒体凋亡途径影响肝癌的可能潜在机制。

具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体的构建

目的构建具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体。方法本研究在前期构建的多表位疫苗GILE的基础上,通过添加SAM基因序列,设计、合成SAM-GILE序列,构建乳酸菌表达质粒pNZ8148-SAM-GILE,并电转至乳酸乳球菌NZ9000中建立乳酸菌表达体系。用酶切与基因测序双重实验验证乳酸菌表达质粒pNZ8148-SAM-GILE是否构建成功,用PCR验证乳酸菌表达质粒pNZ8148-SAM-GILE是否成功电转至乳酸乳球菌NZ9000,用Western Blot实验检测重组蛋白SAM-GILE是否表达,用全细胞ELISA法检测SAM-GILE的表面展示情况,用免疫荧光染色法鉴定LL-plSAM-GILE的M细胞靶向性。结果酶切与基因测序双重实验结果显示,乳酸菌表达质粒pNZ8148-SAM-GILE构建成功。经PCR验证,pNZ8148-SAM-GILE成功电转至乳酸乳球菌NZ9000。经Western Blot验证,通过Nisin诱导,成功表达约45 KD的重组蛋白。经全细胞ELISA检测,SAM-GILE可展示于LL-plSAM-GILE表面。经免疫荧光染色验证,重组抗原与M细胞高度重合,表明重组乳酸菌疫苗LL-plSAM-GILE具有M细胞靶向性。结论成功构建具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体。