CircDOCK7 facilitates the proliferation and adipogenic differentiation of chicken abdominal preadipocytes through the gga‑miR‑301b‑3p/ACSL1 axis

Background Abdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes,which is a well-orchestrated multistep process involving many regulatory molecules.Circular RNAs(circRNAs)have emergingly been implicated in mammalian adipogenesis.However,circRNA-mediated regulation in chicken adipogenesis remains unclear.Our previous circRNA sequencing data identified a differentially expressed novel circRNA,8:27,886,180|27,889,657,during the adipogenic differentiation of chicken abdominal preadipocytes.This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes,and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.Results Our results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7(DOCK7)gene,abbreviated as circDOCK7.CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life.CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens,and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Functionally,the gain-and loss-of-function experiments showed that circDOCK7 promoted proliferation,G0/G1-to S-phase progression,and glucose uptake capacity of chicken abdominal preadipocytes,in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes.Mechanistically,a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p,which was identified as an inhibitor of chicken abdominal adipogenesis.Moreover,the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p.Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator.Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Conclusions CircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene,thus augmenting adipogenesis in chickens.These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.

Protective efficacy of an H5/H7 trivalent inactivated vaccine(H5-Re13,H5-Re14, and H7-Re4 strains) in chickens, ducks, and geese against newly detected H5N1, H5N6, H5N8, and H7N9 viruses

Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains(H5-Re11 and H5-Re12) used in China. In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production. The vaccine strain H5-Re13 contains the hemagglutinin(HA) and neuraminidase(NA) genes of an H5 N6 virus that bears the clade 2.3.4.4 h HA gene, H5-Re14 contains the HA and NA genes of an H5 N8 virus that bears the clade 2.3.4.4 b HA gene, and H7-Re4 contains the HA and NA genes of H7 N9 virus detected in 2021. We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese. The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested. Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses(an H5 N1 virus, an H5 N6 virus, and an H5 N8 virus) bearing the clade 2.3.4.4 b HA gene, an H5 N6 virus bearing the clade 2.3.4.4 h HA gene, and an H7 N9 virus. All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses. Similar protective efficacy against H5 viruses bearing the clade 2.3.4.4 h or 2.3.4.4 b HA gene was observed in ducks and geese. Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7 N9 viruses that are currently circulating in nature.

The Expression of Interleukin-22 and S100A7, A8, A9 mRNA in Patients with Psoriasis Vulgaris

In order to study the expression of interleukin-22 （IL-22） and S 100A7, A8, A9 mRNA in the skin lesions of patients with psoriasis vulgaris and their relationship, the biopsies were taken from skin lesions in 35 patients with psoriasis vulgaris and the skin of 16 normal controls, and the expression levels of 1L-22 and S 100A7, A8 and A9 mRNA were detected by semi-quantitative RT-PCR. The results showed that （1） IL-22 and S 100A8, A9 mRNA were positively expressed in the psoriatic skin lesions but negatively expressed in the normal controls; The expression level of S 100A7 was （1.133±0.040） in the psoriatic skin lesions, significantly higher than that in the normal controls （0.744±0.037, P〈0.01）. （2） There were significantly positive correlations between the expression of IL-22/S100A7 mRNA, IL-22/S100A8 mRNA, IL-22/S100A9 mRNA in the psoriasis vulgaris （r1=-0.543, r2=0.774, r3=0.621, P〈0.01）. It was concluded that IL-22 and S 100A7, A8, A9 might play important roles in the occurrence and progression of psoriasis.

Science Letters:Assignment of CCR7 gene to chicken chromosome 27 by radiation hybrid panel mapping

The protein encoded by CC chemokine receptor 7 (CCR7) is a member of the G protein-coupled receptor family. This receptor was identified as a gene induced by the Epstein-Barr virus (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. This receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. It has been shown to control the migration of memory T cells to inflamed tissues, as well as stimulate dendritic cell maturation. To map the CCR7 gene in chicken chromosome, a 6 000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. PCR of samples from ChickRH6 revealed that the location of CCR7 gene is linked to the maker SEQ0347 (6 cR away) with LOD score of 16.6 and that the marker SEQ0347 is located on chromosome 27 at 27 cR of RH (radiation hydrid) map. We compared the corresponding human mRNA sequence with the predicted coding sequence of chicken CCR7 gene, and found that the assembled contig shared a high percentage of similarity with that of the human gene.

Autocrine Production of Interleukin-6: A Mechanism of Interleukin-6 Independence in Dexamethasone-Resistant 7TD1 Murine Myeloma Cells

Several factors could contribute to proliferation of multiple myeloma (MM) cells independent of interleukin-6 (IL6) in the later stages of the disease. Our previous studies established a dexamethasone-resistant 7TD1 cell line (7TD1-Dxm) and have shown that one mechanism of resistance to dexamethasone is due to inhibition of cytochrome c release. We have also observed that 7TD1-Dxm cells proliferate independently of externally-added IL6. This study therefore aimed to elucidate the mechanisms responsible for IL6-independent proliferation in 7TD1-Dxm cells. Our results indicated that 7TD1-Dxm cells produced IL6 in an autocrine fashion. We have observed that dexamethasone-resistant 7TD1 cells become dexamethasone-resistant and IL6-independent for proliferation concomitantly. This strongly suggests that production of IL6 by 7TD1-Dxm cells may play an important role in the development of dexamethasone resistance. Consequently, further investigation of the molecular mechanisms responsible for IL6 production may be helpful in delineating the mechanisms leading to dexamethasone resistance.

Chicken toll-like receptor 7的研究进展及展望

TLRs(toll-like receptors,TLRs)是一种天然免疫分子,它通过识别病原体相关分子模式在其宿主机体的天然免疫系统起着重要的防御作用。chTLR7(Chicken toll-like receptor 7)和其他Toll家族成员一样属于Ⅰ型跨膜蛋白受体,位于鸡的1号染色体上。本文主要探讨了chTLR7的发现及其配体、分布、转导途径、生物学功能及其与细胞凋亡关系论述,另外对其以后的研究进行了展望进而更进一步了解其功能。

重组鸡白细胞介素-7在HEK293T细胞中的表达及其条件分析

为提高重组鸡白细胞介素-7(chIL-7)在真核细胞中的表达水平,本试验分析了影响基因表达的一些因素,包括转染方法、质粒用量、转染时间及表达时间等,对重组chIL-7表达水平的影响,并通过正交试验设计方法确定了利用HEK293T细胞表达重组chIL-7的最适表达条件。结果表明,在T75细胞瓶中,由脂质体介导使用30μg质粒,转染4h,表达48h和由磷酸钙介导使用40μg质粒,转染6h,表达60h可以获得较高的表达效率,表达水平分别为(124±9)和(94.3±8)μg/106个细胞。本结果为进一步研究chIL-7的功能提供有利条件。

Approach to loss of response to advanced therapies in inflammatory bowel disease

BACKGROUND Remarkable progress over the last decade has equipped clinicians with many options in the treatment of inflammatory bowel disease.Clinicians now have the unique opportunity to provide individualized treatment that can achieve and sustain remission in many patients.However,issues of primary non-response(PNR)and secondary loss of response(SLOR)to non-tumour necrosis factor inhibitor(TNFi)therapies remains a common problem.Specific issues include the choice of optimization of therapy,identifying when dose optimization will recapture response,establishing optimal dose for escalation and when to switch therapy.AIM To explores the issues of PNR and SLOR to non-TNFi therapies.METHODS This review explores the current evidence and literature to elucidate management options in cases of PNR/SLOR.It will also explore potential predictors for response following SLOR/PNR to therapies including the role of therapeutic drug monitoring(TDM).RESULTS In the setting of PNR and loss of response to alpha-beta7-integrin inhibitors and interleukin(IL)-12 and IL-23 inhibitors dose optimization is a reasonable option to capture response.For Janus kinase inhibitors dose optimization can be utilized to recapture response with loss of response.CONCLUSION The role of TDM in the setting of advanced non-TNFi therapies to identify patients who require dose optimization and as a predictor for clinical remission is not yet established and this remains an area that should be addressed in the future.

传染性腔上囊炎疫苗免疫鸡TLR7基因表达的动态变化

给11日龄雏鸡分别口服接种鸡传染性腔上囊炎(IBD)弱毒疫苗(法贝灵,IBD-Blen)1和3头份后,于不同时间采集脾和腔上囊组织,分离纯化淋巴细胞,常规方法抽提总RNA,以鸡β-actin基因为内参,采用半定量RT-PCR法检测两种组织中鸡Toll样受体7(ChTLR7)基因的表达动态。结果显示,接种IBD弱毒疫苗前,试验鸡脾和腔上囊组织中均有ChTLR7的微量表达,疫苗接种后第7 h,脾中ChTLR7 mRNA的表达开始显著上调,至接种后第12 h达峰值,此后迅速下降,至第130 h时降至疫苗接种前的水平;而腔上囊组织中ChTLR7 mRNA的表达自接种后第12 h开始增加,第24 h时达峰值,随后迅速下降,约72 h后恢复至疫苗接种前水平。表明ChTLR7可能参与了IBDV感染早期的天然免疫反应过程。

不同致病性H9N2亚型禽流感病毒诱导鸡TLR-7和Mx基因mRNA转录水平的差异

本试验拟研究不同致病性H9N2亚型禽流感病毒诱导SPF鸡、SPF鸡胚和鸡胚成纤维细胞中TLR-7和Mx基因mRNA转录水平的动态变化,为不同致病性H9N2亚型禽流感病毒诱导IFN-Ⅰ产生差异的机制提供依据。150只6周龄SPF鸡随机分为3组,分别静脉接种PBS、105 EID50毒株SD/196和SD/818各0.2mL,分别于接种后的第1、3、5、7和10天每组剖杀8只,收集具有明显病变差异的组织;180枚10日龄SPF鸡胚分别尿囊腔接种PBS、104 EID50的SD/196毒株和SD/818毒株各0.1mL,分别于接种后的第4、8、16、24、32和48小时每组收集胚体10个;鸡胚成纤维细胞分别接种PBS、SD/196和SD/818毒株病毒液(1moi)进行37℃培养,于感染后的第4、8、16、24、32和48小时收集细胞;动态收集的病变组织、胚体和成纤维细胞通过荧光定量PCR测定TLR-7和Mx基因mRNA水平的表达。结果显示,肺、肾、十二指肠和法氏囊为主要病变差异组织,除鸡胚TLR-7的表达外,毒株SD/818大部分时间点诱导主要病变差异组织、鸡胚和成纤维细胞中TLR-7和Mx基因mRNA的表达显著高于毒株SD/196(P<0.05)。结果表明不同致病性H9N2亚型禽流感病毒明显诱导了SPF鸡主要病变差异组织、SPF鸡胚和鸡胚成纤维细胞中TLR-7和Mx基因mRNA的表达,且较高致病性毒株SD/818诱导表达量明显高于较低致病性毒株SD/196。

不同放养密度对雪峰乌骨鸡生长性能、屠宰性能、血清生化和免疫指标的影响

本试验旨在研究不同放养密度对雪峰乌骨鸡生长性能、屠宰性能、血清生化以及免疫指标的影响。选用健康的35日龄、体重一致的脱温雪峰乌骨鸡母鸡苗1620只,随机分为3个放养密度组,分别为低密度组(LSD组,60只/667 m^(2))、中密度组(MSD组,120只/667 m^(2))和高密度组(HSD组,180只/667 m^(2)),每组6个重复(林地面积500 m^(2)),LSD组每个重复45只,MSD组每个重复90只,HSD组每个重复135只。预试期7 d,正试期78 d。结果显示:1)LSD组雪峰乌骨鸡的平均日增重和料重比显著高于MSD组和HSD组(P<0.05)。2)LSD组宰前活重和屠体重均显著高于MSD组和HSD组(P<0.05)。3)LSD组血清中谷草转氨酶活性显著低于HSD组(P<0.05),LSD组和MSD组血清中碱性磷酸酶活性显著高于HSD组(P<0.05);LSD组血清中甘油三酯含量显著高于HSD组(P<0.05);HSD组血清中超氧化物歧化酶活性显著低于LSD组和MSD组(P<0.05)。4)HSD组血清中免疫球蛋白G、免疫球蛋白M、白细胞介素-2、白细胞介素-6和肿瘤坏死因子-α含量显著高于LSD组和MSD组(P<0.05),MSD组血清中免疫球蛋白A含量显著高于LSD组和HSD组(P<0.05)。由此可见,放养密度为60只/667 m^(2)时能有效提高雪峰乌骨鸡的生长性能,同时保证有较好的屠宰性能、抗氧化能力及免疫力。

鸡BMP7基因组织表达特性及其在高、低脂系脂肪组织中表达差异研究

骨形态发生蛋白7(Bone morphogenetic proteins7,BMP7)是一类与骨骼发育密切相关的酸性多肽,属于TGF-β超家族,能够诱导血管周围及结缔组织中的间充质细胞向骨细胞方向分化。近期研究结果表明,BMP7在哺乳动物棕色脂肪组织发育过程中发挥重要作用。目前还没有关于鸡BMP7基因表达规律的研究。本研究以东北农业大学高脂系肉鸡为试验材料,采用半定量RT-PCR的方法检测BMP7基因在不同组织中的表达特性,发现BMP7在鸡23种组织中均有表达;采用半定量RT-PCR和Real-time PCR的方法检测BMP7基因在高、低脂系肉鸡脂肪组织间的表达差异,发现BMP7基因在高脂系肉鸡脂肪组织中的表达量显著高于低脂系(P<0.05)。本研究为进一步揭示BMP7基因与鸡脂肪组织发育之间的关系奠定了基础。

鸡IRF7原核表达及多克隆抗体制备

为了制备鸡干扰素调节因子7(interferon regulatory fator 7,IRF7)抗体,本研究通过分离鸡的外周血淋巴细胞,抽提RNA并反转录获得鸡淋巴细胞c DNA,利用聚合酶链式反应扩增获得鸡IRF7基因(ch IRF7),将该基因克隆入原核表达载体p COLD-TF中,构建成p COLD-TF-ch IRF7原核表达质粒。将测序正确的表达质粒转化入大肠杆菌BL21(DE3)感受态中,经0.25 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)低温诱导20 h。SDS-PAGE结果显示,融合蛋白获得高效表达,大小为107 k Da。切胶免疫BALB/c小鼠,制备鼠抗鸡IRF7多克隆抗体。激光共聚焦、Western blot实验结果显示,该抗鸡IRF7多克隆抗体能与真核质粒表达的ch IRF7蛋白发生特异性反应。该抗体的成功制备为进一步研究鸡IRF7的表达调控机制奠定了基础。

鸡柔嫩艾美尔球虫第二代裂殖子表面抗原MZ5-7基因的克隆、表位分析及原核表达

用RTPCR的方法,从感染柔嫩艾美尔球虫(Eimeriatenella)第5天的鸡盲肠分离的第二代裂殖子中克隆出MZ57基因,并进行抗原表位分析和原核表达,结果表明,MZ57基因ORF为945bp,与GenBank里已知序列(登录号为L08257)比较核甘酸和氨基酸同源性均为99.9%。应用DNAStar软件对该基因的抗原表位进行了分析,发现其B细胞表位广泛分布在80～300区域,其中80～130区域、190～220区域和300～320区域更为突出,而T细胞基序则集中在123～190区域和220～300区域。根据MZ57基因序列重新设计一对引物,PCR方法扩增出不含信号肽长度为885bp的基因序列,克隆到pET28b获得重组质粒pET28bMZ亚,转入宿主菌BL21中,用IPTG进行诱导表达,表达的融合蛋白约36.5kDa10poundnote,符合目的蛋白大小;同时应用Westernblot方法与抗第二代裂殖子和子孢子的2种高免血清反应,结果表明抗裂殖子和子孢子血清均能识别MZP(裂殖子蛋白,MZP,merozoiteprotein)。

Oncolytic adenovirus-mediated MDA-7/IL-24 overexpression enhances antitumor activity in hepatocellular carcinoma cell lines

BACKGROUND: Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells. We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24, both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2, Hep3B, SMMC-7721, HCCLM3, and the normal liver cell line L02. METHODS: Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24. The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR, ELISA, and Western blotting. MTT assay was used to investigate the proliferation effect. Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line. Flow cytometry was used to analyse the cell cycle. RESULTS: RT-PCR, ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression, promoted apoptosis, and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS: SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24, SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines. (Hepatobiliary Pancreat Dis Int 2010; 9:615-621)

Role of P2X_7 receptors in the development of diabetic retinopathy

The P2X7 receptor is one of the members of the family of purinoceptors which are ligand-gated membrane ion channels activated by extracellular adenosine 5'-triphosphate. A unique feature of the P2X7 receptor is that its activation can result in the formation of large plasma membrane pores that allow not only the flux of ions but also of hydrophilic molecules of up to 900 Da. Recent studies indicate that P2X7-mediated signaling can trigger apoptotic cell death after ischemia and during the course of certain neurodegenerative disorders. Expression of the P2X7 receptor has been demonstrated in most types of cells in the retina. This purinoceptor mediates the contraction of pericytes and regulates the spatial and temporal dynamics of the vasomotor response through cell-to-cell electrotonic transmission within the microvascular networks. Of potential clinical significance, investigators have found that diabetes markedly boosts the vulnerability of retinal microvessels to the lethal effect of P2X7 receptor activation. This purinergic vasotoxicity may result in reduced retinal blood flow and disrupted vascular function in the diabetic retina. With recent reports indicating an association between P2X7 receptor activation and inflammatory cytokine expression in the retina, this receptor may also exacerbate the development of diabetic retinopathy by a mechanism involving inflammation.

KLF7通过抑制缺氧诱导因子1α转录影响肉鸡脂肪组织发育

Krüppel样转录因子7(Krüppel-like factor 7,KLF7)是脂肪形成的负调控因子,而缺氧诱导因子1(hypoxia-inducible factor 1,HIF1)促进缺氧诱导的哺乳动物脂肪组织发育。本实验室前期利用ChIP-seq技术,发现在鸡的缺氧诱导因子1α(hypoxia-inducible factor 1 alpha,HIF1α)基因上游存在1个KLF7的结合峰,提示KLF7可能调控HIF1α基因转录。为此,本研究首先利用ChIP-PCR技术验证ChIP-seq结果,发现KLF7能够与HIF1α基因5′侧翼区结合。双荧光素酶报告基因与qRT-PCR结果显示,过表达KLF7能够显著下调HIF1α(-4432/-4182)的荧光素酶报告基因活性(P<0.01),抑制HIF1α基因表达。与野生型质粒相比,将生物信息学预测的KLF7结合基序"TGCGCAGCAA"(-4300/-4290)缺失突变后,HIF1α(-4432/-4182)报告基因活性显著增强(P<0.01)。此外,选取第19代1~7周龄东北农业大学高、低脂双向选择品系肉鸡(Northeast Agricultural University broiler lines divergently selected for abdominal fat content,NEAUHLF)作为实验材料,利用qRT-PCR检测HIF1α基因在肉鸡腹部脂肪组织中的表达规律。结果显示,HIF1α在1~7周龄高脂系肉鸡中的相对表达量均高于低脂系,提示HIF1α对鸡脂肪组织发育具有促进作用。在永生化鸡前体脂肪细胞系(immortalized chicken preadipocyte cell line,ICP1)诱导分化过程中,HIF1α相对表达量逐渐升高。综上所述,HIF1α是转录因子KLF7的一个靶基因,KLF7可能通过抑制HIF1α基因转录参与鸡脂肪组织发育过程。

Dramatic Changes of Matrix Metalloproteinases-7 and Lysozyme in the Ulcerative Colitis of Mice Induced by Dextran Sulfate Sodium

Ulcerative colitis （UC） is a lifelong illness with profound emotional and social impacts, and could cause serious damage to large intestine, especially in colon. However, the pathogenesis of UC remained unclear. The present study attempts to find out the role of matrix metalloproteinases-7 （MMP-7） and lysozyme in the pathogenesis of UC through a mice model induced by dextran sulfate sodium （DSS）. The UC model was evaluated both by disease activity index （DAI） and the intestinal histopathology. The results show that there is a high correlation between the DAI score and the pathological changes of colon. Interleukin-6 （IL-6） serum levels and large intestinal fluids levels in UC mice are always higher than that of the control groups, which might be associated with the degree of the inflammation damage in the colon. The change tendency of the MMP-7 mRNA and protein expressions are both up-regulated firstly and then down-regulated from 1 to 5 d in the colon, but only the MMP-7 protein is up-regulated at 7 d again. The up-regulated MMP-7 levels in the early stage of UC may play a protective role through the activated defensins, while the down-regulated levels in the mid-later stage of UC may be connected with the severe lesions in the colon. However, the up-regulated MMP-7 levels in the later stage of UC in the colon may also contribute to the tissue repair or be served as a marker to CRC （colorectal cancer）. The distribution of lysozyme protein indicates that there may be Paneth-like cells in the colon. Both the changes of MMP-7 and lysozyme in the small intestine may play a protective role for the safe environment of the whole gut, especially to the colon of UC.

Differential Regulation of Cytochrome C Release in Dexamethasone-Resistant 7TD1 Cells

Interleukin-6 (IL6)-triggered JAK/STAT3 and PI3K/AKT signaling pathways are known to mediate cell survival, drug resistance and progression in a variety of cancer cells. Resistance to induction of apoptosis plays a critical role in the pathogenesis of numerous cancers and development of resistance to chemotherapeutic agents used in its treatment. Previous research in our laboratory employing a dexamethasone-resistant subline (7TD1-Dxm) of IL6-dependent 7TD1 cells indicated that constitutively activated STAT3 was important in control of apoptosis and targets downstream to activated STAT3 appeared to be involved in the development of resistance to dexamethasone by 7TD1 cells. We therefore investigated the hypothesis that Dxm-resistance developed by 7TD1-Dxm cells was due to resistance to induction of apoptosis mainly because of the dysregulation of the downstream targeted in JAK/STAT3 signaling pathway. Our results indicate that 7TD1-Dxm cells show resistance to Dxm-induced reduction of Bcl-2 protein and the release of cytochrome c. Thus, this study suggests that development of resistance to dexamethasone by 7TD1 cells may involve altered regulation of mitochondrial anti-apoptotic proteins.

鸡骨骼肌卫星细胞系的建立及分析

旨在通过慢病毒包装SV 40-LT基因并转导PMSCs以构建鸡骨骼肌卫星细胞系。本研究采集20~30枚15胚龄健康AA鸡的胸部组织,采用混合酶消化法分离培养PMSCs。将细胞随机分为两组,每组3个重复,处理组选择SV 40-LT慢病毒转染48 h后,更换含1μg·mL^(-1)的嘌呤霉素筛选培养基于处理组和对照组(病毒不转染)。待对照组细胞全部死亡,对处理组细胞进行不断传代培养,最终获得鸡骨骼肌卫星细胞系。免疫荧光试验分析鸡骨骼肌卫星细胞标志基因PAX7的表达;采用CCK-8和细胞周期分析检测细胞增殖特性;血清依赖性和软琼脂分析检测其恶性转化情况;构建诱导分化模型检测其诱导分化能力。结果表明,PMSCs中有92%细胞PAX 7基因呈阳性,可用于后续研究。SV 40-LT基因在鸡骨骼肌卫星细胞系(IMSCs P12)中的表达是原代鸡骨骼肌卫星细胞的15倍,且连续传代至12代后,IMSCs P12与PMSCs均呈纤维样状。IMSCs P12具有更高的增殖活性且没有发生恶性转化,随后,对IMSCs P12进行诱导分化,在其增殖至70%到诱导分化1 d这一阶段,IMSCs P12与PMSCs的PAX 7基因表达下调,而MyoD和MyHC基因表达上调。结果提示,本研究通过转染SV 40-LT基因成功培养了鸡骨骼肌卫星细胞系,其具有与原代鸡骨骼肌卫星细胞相似的特性。该细胞系的建立为研究家禽骨骼肌相关的功能基因提供了新的平台,也为SV 40-LT基因在家禽细胞永生化的研究奠定了基础。