Is type Ⅰ alpha 2 collagen gene responsible for intracranial aneurysm in Northeast China?

In this study, we investigated whether a single nucleotide polymorphism （rs42524 G 〉 C） in the type I alpha 2 collagen gene was associated with sporadic ruptured intracranial aneurysm or its clinical characteristics in patients from Northeast China. Genotyping of the rs42524 G 〉 C polymorphism was carried out using a polymerase chain reaction-restriction fragment length polymorphism assay. The data showed that the frequency of the rs42524 GC ＋ CC genotype was significantly higher than the GG genotype among intracranial aneurysm patients whose Hunt and Hess grading scale was 〉 3. In addition, the rs42524 G 〉 C genotype was found to have a statistically significant association with intracranial aneurysm risk. These findings indicate that the type I alpha 2 collagen gene gene may be involved in a predisposition to intracranial aneurysm in the Northeast Chinese population. Crucially, the rs42524 C allele may be an important risk factor for increased severity of the condition in patients with ruptured intracranial aneurysms.

Ceruloplasmin or Fibronectin Synergism with Quartz Dust on Stimulating Collagen Gene Transcription in Human 2BS Fibroblast

Human α1(Ⅰ), α2(Ⅰ) and α1(Ⅲ) cDNA probes and RNA dot hybridization were employed to quantitate collagen mRNA changes after adding silica dust into the media of human 2BS fibroblasts. At all dosages used (100, 200, 500 and 1000μg), the α1(Ⅰ), α2(Ⅰ)and α1(Ⅲ) mRNA levels increased one day after dusting. At the same dosage of silica (100μg), α1(Ⅲ) mRNA increased earlier than type Ⅰ collagen mRNA did. The type Ⅰ and type Ⅲ collagen mRNA contents in the experimental groups were higher than those in control on days 3, 5, 7 and 9. The effect of ceruloplasmin (Cp) and fibronectin (Fn) on collagen mRNA synthesis was also studied, after adding silica dust, Cp or Fn into the media of human 2BS fibroblast. The results showed that Cp and Fn have stimulating effect on collagen mRNA production. When both Cp and silica dust were added into cell culture media, the collagen mRNA level was increased more than those of adding either Cp or silica dust alone. Similar situations were found for Fn. Cp (or Fn) synergism with silica dust on stimulating transcription of human collagen gene was suggested

Inhibition of α_1(Ⅰ) collagen gene in vitro transcription by antisense oligodeoxynucleotides

Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.

Dexamethasone down- regulates the activity of promoter from human a1(I) procollagen gene

Objective To investigate the effects of dexamethasone on the promoter activity of human a1(I) procollagen gene. Methods Fibroblasts from human skin were primary cultured and subcultured. (1)The effects of dexamethasone on the human skin fibroblasts were determined by BrdU incorporation into DNA of fibroblasts. (2)Three plasmids containing various lengths of 5’flank sequence of human a1(I) procollagen gene and CAT as reporter gene were constructed, and were transfected into the human skin fibroblasts by FuGENE Transfection Reagent. The effects of dexamethasone on 3 plasmids were determined by CAT－ ELISA. Results (1)After 24h of treatment on the fibroblasts with 110－ 9～ 110－ 4mol/L dexamethasone in DMEM containing 2％ or 10％ FCS, BrdU incorporation into DNA showed no difference (P >0.05). (2) the 3 plasmids were transfected into fibroblasts and then treated with 110－ 5mol/L and 110－ 6mol/L dexamethasones for 24h, relative CAT values were different betwent dexamethasone and control, higher dexamethasone(110－ 5mol/L) and lower dexamethasone(110－ 6mol/L) (P< 0.05). Conclusion Dexamethasone has no effects on the proliferation of human skin fibroblasts, and it has negative effect on the promoter activity of human a1(I) procollagen gene, which is dose－ dependent.

Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation,collagen synthesis and gene expression

AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10-4mol/L-10-7mol/L SA-A for 22 h.The cell viability wasassayed by[3H]proline incorporation,cellproliferation by[3H]TdR incorporation,cellcollagen synthetic rate was measured with[3H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10-4mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10-5mol/L-10-7mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10-6mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10-5mol/L-10-6mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10-5mol/L and10-6mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.

指长比(2D∶4D)与胶原蛋白基因家族单核苷酸多态性的关联性

目的:探讨指长比(2D︰4D)与胶原蛋白基因家族4个基因COL4A2、COL5A1、COL6A2、COL10A1的10个单核苷酸多态性位点(rs3809346、rs3825490、rs7984100、rs1134170、rs3128575、rs1042917、rs2839110、rs3812111、rs1064583、rs2228547)的关联性。方法:采用体质测量法和多重PCR法,分别对2D︰4D及COL4A2、COL5A1、COL6A2和COL10A1基因多态性在宁夏地区大学生的分布特征进行比较,并分析其间的关联性。结果:宁夏女性大学生双手2D︰4D均值均显著高于男性;10个位点基因型和等位基因分布频率性别间均无显著差异;COL4A2基因rs3825490和rs3809346位点基因型分别与男性和女性双手2D︰4D显著关联,COL5A1基因rs1134170位点基因型和COL6A2基因rs1042917位点基因型分别与女性左手2D︰4D和男性右手2D︰4D显著关联。结论:宁夏人群2D︰4D的形成可能与COL4A2(rs3809346、rs3825490)、COL5A1(rs1134170)及COL6A2(rs1042917)基因多态性有关。

通过生物信息学分析鉴定糖尿病视网膜病变的关键生物标志物和免疫浸润

目的:通过生物信息学方法为糖尿病视网膜病变中的相关机制提供理论依据。方法:通过GEO数据库筛选出增殖性糖尿病视网膜病变(PDR)数据集GSE60439和GSE94019。使用R语言提取GSE60439数据集中的表达数据进行t检验,筛选出差异基因,筛选条件为P<0.05和|log2FC|≥1;采用WGCNA选择GSE94019数据集中与PDR相关的重要模块;对差异基因及重要模块中的基因取交集得到关键基因。对关键基因进行GO及KEGG富集分析;构建PPI网络鉴定出高风险关键蛋白。最后,使用CIBERSORTx分析免疫细胞在PDR中的浸润,采用Pearson相关性检验分析免疫细胞与关键基因的相关性。结果:最终筛选出3个核心基因,即Col1a1、Col1a2、Col3a1,其表达与M2巨噬细胞呈正相关,且核心基因富集于AGEs-RAGE信号通路。结论:正反馈可能存在于M2巨噬细胞、AGEs-RAGE信号通路及胶原基因(Col1a1、Col1a2、Col3a1)之间,即使没有高糖的刺激,这种恶性循环仍可能持续损害视网膜组织。

Ⅰ型胶原编码基因突变致成骨不全症动物模型

成骨不全症(osteogenesis Imperfecta,OI)是一类以低骨量、骨脆性及骨骼畸形为特征的单基因遗传性骨病,研究其病理生理学机制和有效治疗方法的关键是动物模型的应用。绝大部分OI由编码Ⅰ型胶原相关基因突变引起,本文总结了COL1A1和COL1A2突变的主要动物模型,这些模型是研究致病机制、开发和测试新的治疗策略的宝贵工具。未来的研究将运用CRISPR/Cas9等新型基因编辑技术,结合多种生物类别,优化和拓展OI动物模型,更好地模拟人类疾病,有助于对OI及其治疗方法的深入探索。

胶原蛋白水解物缓解去势小鼠骨质疏松作用机理的探究

胶原蛋白水解物被认为拥有促进成骨和成血管以及起到防止骨质疏松的作用。我们通过生物信息学和建立去势小鼠骨质疏松症模型探究了胶原水解物(CH)对骨质疏松症有益作用的潜在机制。本研究发现,CH可以使24个与骨质疏松和成血管相关基因差异表达,差异基因的富集分析确定了20个KEGG通路。通过对筛选的十个通路进行分析,并结合STITCH数据库的探究,发现了Kdr、Erbb2、Wnt3a、Jagl、Fltl为CH调控的中枢基因。特别是,MAPK信号通路被确定为共享通路中的潜在关键通路。这个研究结果表明,胶原蛋白水解物可以通过MAPK信号通路促进成血管并抑制骨质疏松。

表达元件优化促进重组胶原蛋白在谷氨酸棒杆菌中的表达

重组胶原蛋白是一种具有广泛应用潜力的生物材料,近年来引起了生物医学、组织工程等众多领域的关注。胶原蛋白的3股螺旋结构赋予其独特的生物学功能和生物相容性,但也增加了其在微生物系统中表达的复杂性。该研究以细菌胶原蛋白V-B作为模式蛋白,通过对表达元件的优化,促进重组胶原蛋白在谷氨酸棒杆菌中的表达。首先通过启动子筛选和发酵时长优化,获得介导V-B表达的最优启动子tac-R0。接着利用RBS calculator设计核糖体结合位点(ribosomal binding site,RBS)和间隔序列(aligned spacing,AS)的突变文库,得到与tac-R0启动子搭配组合的最优RBS和AS,使V-B的产量提高至514 mg/L。此外,通过多基因表达盒的串联组合策略,将多个目的基因串联,最终V-B的产量较初始水平提高了8.4倍,达697 mg/L,该研究为重组胶原蛋白的工业化生产提供了基础。

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor（β-NGF） on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS（β-NGF ＋ PBS） into the right-hand side defect, and PBS into the left（control） defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF ＋ PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF ＋ PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

Genomic Analysis of 727 Patients with Ehlers-Danlos Syndrome I: Clinical Perspective Relates 23 Genes to a Maternally Influenced Arthritis-Adrenaline Disorder

A novel medical approach for qualifying DNA variants found by whole exome sequencing (WES) facilitates discovery of new gene-disease relationships and emphasizes that DNA change must be correlated with clinical findings before having utility for diagnosis. Delineation of an arthritis-adrenaline disorder (AAD) process qualified variants in 23 genes as diagnostically useful in 727 patients having WES among 1656 with Ehlers-Danlos syndrome (EDS);these results distinguished them from 102 patients who had qualified gene variants among 728 with developmental disability. Excess maternal transmission of AAD by pedigree analysis plus 167 maternally versus 111 paternally transmitted DNA variants and 75 patients with only mitochondrial DNA variants suggest maternal influence on inheritance of AAD and its subsumed EDS types. Genes grouped by impact on different connective tissue elements showed variation in similar numbers of patients with hypermobile or classical EDS, benign joint hypermobility, or predominant dysautonomia: COL7A1, FLG acting on skin in 21 patients;SCN9A/10A/11A, POLG on nerve in 24;COL6A1/A2/A3, COL12 on muscle in 19;COL5A1/A2, FBN1, TGFB2/3, TGFBR1/2 on tissue matrix in 51;COL3A1, VWF on vessel in 18;COL1A1/A2, COL11A1/A2 acting on bone in 15 patients. Each gene group acts through a postulated articulo-autonomic dysplasia cycle to produce reciprocal tissue laxity and dysautonomia findings that transcend EDS types. This same tissue laxity-dysautonomia cycle acts to produce secondary complications in disorders ranging from distinctive connective tissue dysplasias to developmental disorders with hypotonia and acquired conditions with autonomic imbalance. Several altered genes were previously associated with neuromuscular disorders, foreshadowing a large myopathic EDS category that will incorporate many patients with hypermobility. The importance of muscle for joint constraint supports present exercise and future mesenchymal stem cell therapies, whether AAD is genetic or epigenetic from trauma, surgery, inflammation, or aging.

LOCALIZATION OF TYPE I AND TYPE Ⅲ PROCOLLAGEN mRNAs IN BREAST SCIRRHOUS CARCINOMA BYIN SITU HYBRIDIZATION

Scirrhous carcinoma is charactertzed by reinarkable amount of collagen fibrils, mainly type I and type III collagens. The origin of collagens is still under debate.cDNA fragments of type I and type III procollagens were subcloned into Gemini pGEM vectors to synthesize the 35Slabeled cRNA probes. By in situ hybridization, we have found the fibroblasts surrounding the tumor cells and cords contained abundent type I and type III procollagen mRNAs which decreased with the distance of fibroblasts from the tumor cells. In all freshly prepared tissues, the tumor cells also contained significant pro α1 (I) and pro α1 (III) mRNAs, but no or little pro α2 (I) mRNA. The results indicated that type I and type III collagens in human scirrhous carcinoma of breast are mainly produced by fibroblasts. Tumor cells also perticipate in the disposition of collagen fibrils, probably type I trimer and type III collagens in accordance with what was observed in biochemical

COL1A1、MMP3基因多态性与中国青年男性力量素质相关血液指标的关联研究

目的探讨α-I型胶原(α-type I collagen,COL1A1)基因rs1107946位点、基质金属蛋白酶(matrix metalloproteinases 3,MMP3)基因rs650108位点多态性与长期坚持运动的青年男性体能素质的关联性。方法选取374名青年男性,测量体成分,测试体能,同时,采集血样检测血常规、血液生化指标及基因位点多态性。结果COL1A1基因rs1107946位点不同遗传模式CC基因型跪推实心球成绩高于AC基因型(P=0.037)、AA基因型(P=0.028)及AC+AA基因型(P=0.013);CC基因型俯卧撑成绩高于AC基因型(P=0.046)、AC+AA基因型(P=0.034);CC基因型立定跳远成绩高于AA基因型(P=0.006),C等位基因型(AC+CC)立定跳远成绩显著高于AA基因型(P=0.011)。MMP3基因rs650108位点不同遗传模式AA基因型跪推实心球成绩高于GA基因型(P=0.023)及GA+GG基因型(P=0.024)。COL1A1基因rs1107946位点CC基因型较AA基因型和AC基因型血清TBil和DBil高水平(P=0.040,P=0.041)、ALP低水平(P=0.018)。MMP3基因rs650108位点AA基因型较GG基因型和GA基因型HCT高水平(P=0.045),PCT、HDL-C低水平(P=0.011,P=0.012)。结论COL1A1 rs1107946、MMP3 rs650108基因多态性可作为力量素质的分子遗传标记,COL1A1 rs1107946 CC基因型和MMP3 rs650108 AA基因型是力量素质的优势基因型。

Inhibition of TGF-β1 by eNOS gene transfer provides cardiac protection after myocardial infarction

Objective： Endothelial nitric oxide synthase （eNOS） and nitric oxide （NO） have been implicated in protection against myocardial ischemia injury. This study was designed to explore a new method of therapy for myocardial injury by eNOS gene transfection. Methods： A rat model of myocardial infarction （MI） was established by left anterior descending （LAD） coronary artery ligation, eNOS gene in an adenovirus vector was delivered locally into the rat heart and hemodynamic parameters were examined after 3 weeks, Matrix metalloproteinase-2 and 9 （MMP-2, MMP-9） mRNA were measured by reverse transcription polymerase chain reaction （RT-PCR）, and the protein levels of eNOS, caspase-3, and transforming grouth factor 131 （TGF-131） were determined by western blot assay. Results： eNOS gene transfer significantly reduced cardiomyocyte apoptosis and improved cardiac function. In addition, eNOS significantly reduced the mRNA levels of MMP-2 and MMP-9. In the eNOS gene transfected group, the activation of caspase-3 and TGF-β1 were decreased. However, the protection was reversed by administration of the NOS inhibitor, N（o））-nitro-l-arginine methyl ester （L-NAME）. Conclusion： These results demonstrate that the eNOS provides cardiac protection after myocardial infarction injury through inhibition of cardiac apoptosis and collagen deposition, and suppression of TGF-β1.

重组胶原蛋白表达体系研究进展

胶原蛋白是哺乳动物中含量最多的蛋白质,至今已发现28种类型,主要分为纤维性胶原蛋白、网状胶原蛋白、珠状丝状胶原蛋白、锚定纤维蛋白、膜蛋白以及multiplexins胶原蛋白,其中纤维性胶原蛋白中Ⅰ型、Ⅱ型、Ⅲ型胶原蛋白占人体胶原蛋白的80%~90%。目前,根据来源,胶原蛋白大致分为动物源胶原蛋白和重组胶原蛋白。动物源胶原蛋白主要来源于陆生动物以及海洋动物,而重组胶原蛋白是指将人胶原蛋白基因克隆到选定的表达载体并转化到表达细胞内,最后通过纯化技术所获得的蛋白质。本文简述了胶原蛋白的结构、类别和生物合成机制,重点阐述了重组胶原蛋白表达体系及特点,包括原核生物、酵母、植物、杆状病毒以及哺乳动物细胞等表达体系及其优势与局限性,介绍了重组胶原蛋白市场前景及在眼科、软骨工程、皮肤治疗等生物医药方面的实际应用,并对重组胶原蛋白的研究和产业发展进行了展望。

骨质疏松受体基因研究方向

骨质疏松症(osteoporosis)是一种以骨量低下、骨组织微结构损坏,导致骨脆性增加,易发生骨折为特征的全身性骨病。骨质疏松是多因素、多基因疾病,是遗传和环境因素交互作用的结果。笔者论述了降钙素受体基因、甲状旁腺素受体基因、雌激素受体基因、肿瘤坏死因子基因、胰岛素样生长因子1受体基因、骨保护素基因、转化生长因子基因、维生素D受体基因、Ⅰ型胶原蛋白基因的生物学特征及其与骨密度、骨质疏松、骨质疏松性骨折的关联,为骨质疏松的发病机制、预防治疗、新药开发研究提供分子生物学依据。

糖皮质激素在受体介导下抑制胶原编码基因表达引发软骨成骨损伤机制

目的研究糖皮质激素诱导软骨损伤的内在机制。方法选用3种不同浓度的经典糖皮质激素泼尼松龙处理斑马鱼幼鱼,用同样量的二甲基亚砜(0.01%)处理同胞幼鱼作为对照组。在第4、5和6天采集处理后的样本进行软骨染色实验;采集6 d后的样本进行qRT-PCR分析;使用JASPAR在线软件预测和分析6个胶原编码基因的糖皮质激素反应元件,并构建由6个胶原编码基因组成的启动子驱动的荧光素酶报告载体,将其与糖皮质激素受体(glucocorticoid receptor,GR)和/或糖皮质激素泼尼松(prednisone,PN)转染到细胞中,测量荧光素酶活性。结果PN 25μmol/L浓度对斑马鱼软骨造成明显损伤。随着PN处理浓度的增加,胶原蛋白编码基因相对mRNA水平显著降低。单次转染GR后,除col10a1a外,所有胶原编码基因的荧光素酶活性均降低;单次添加PN后,6个基因的荧光素酶活性均降低;当GR和PN同时添加时,荧光素酶活性显著降低。结论PN可引起明显的斑马鱼软骨发育损伤,胶原蛋白编码基因表达降低是PN引起斑马鱼软骨损伤的原因,而PN通过GR抑制胶原编码基因的表达。

COL10A1基因新突变导致Schmid型干骺端软骨发育不全

目的对2021年1月陆军军医大学第二附属医院接诊的1例Schmid型干骺端软骨发育不全(Schmid type metaphyseal chondrodysplasia,SMCD)家系进行疾病表型与基因型分析,结合文献复习探讨其防治手段。方法对先证者进行家系调查及系谱分析,收集先证者及家系成员的临床资料,采用全外显子组测序明确突变基因,并通过Sanger测序验证和家系共分离分析,同时结合ACMG指南进行生物信息学分析来评价变异的致病性。结果在家系患者中发现COL10A1基因存在新的杂合错义突变c.1843T>G(p.Tyr615Asp),该位点所在区域在不同物种之间高度保守,此突变可能通过影响X型胶原(α1)蛋白的三聚化及其与细胞外基质分子结合导致疾病发生。结论发现了1种SMCD的新突变,丰富了SMCD患者的突变谱,为SMCD的预防、诊断及治疗提供理论依据。

胶原蛋白海绵对大鼠拔牙后牙槽窝愈合过程的影响

目的:探讨胶原蛋白海绵对拔牙创牙槽窝愈合过程的影响。方法:取健康雌性SD大鼠16只,首先建立大鼠拔牙模型,实验侧(右)牙槽窝内填入胶原蛋白海绵,对照侧(左)作空白处理。于拔牙术后1、2、4、8周处死大鼠,观察牙槽窝成骨情况,利用实时定量PCR检测实验侧与对照侧成骨相关基因表达变化。采用SPSS 19.0软件包进行统计学分析。结果:术后实验侧牙槽窝较对照侧愈合明显好,实验侧及对照侧组织中Osterix、Runx2及Vegf均有表达,实验侧组织相关基因在术后1、2、4、8周不同时间点的表达水平显著高于对照侧(P<0.05)。结论:胶原蛋白海绵填塞拔牙创牙槽窝可在一定程度上促进牙槽窝早期骨愈合,这一过程可能与胶原蛋白海绵调节成骨相关基因表达水平有关。