大肠杆菌感染对绵羊乳腺成纤维细胞损伤及TLR4表达的影响

doi:10.7668/hbnxb.20192742

摘要/Abstract

摘要：

为研究绵羊大肠杆菌性乳腺炎中乳腺成纤维细胞的损伤情况及TLR4的作用机制,通过体外培养获得原代绵羊乳腺成纤维细胞,大肠杆菌人工感染细胞建立大肠杆菌性乳腺炎细胞模型,分析大肠杆菌对细胞损伤的影响及TLR4在大肠杆菌性乳腺炎中的作用。采用酶消化法结合差速消化法获得原代绵羊乳腺成纤维细胞;MTT法检测细胞活力及TLR4阻断剂CLI-095的细胞毒性作用;乳酸脱氢酶法检测大肠杆菌对绵羊乳腺成纤维细胞的损伤情况并筛选最适MOI比值;qRT-PCR法检测caspase-3、TLR4的mRNA相对表达量及CLI-095的阻断效果;比色法检测细胞焦亡蛋白caspase-4,5 的酶活性;WB检测TLR4蛋白的相对表达量;平板活菌计数法检测CLI-095对大肠杆菌黏附绵羊乳腺成纤维细胞的影响。分离获得绵羊乳腺成纤维细胞,MTT测定结果显示,细胞活力良好;大肠杆菌以最适MOI=4:1感染绵羊乳腺成纤维细胞后,各时间段 LDH 释放量均显著升高;caspase-3 mRNA的相对表达量在感染1~3 h内显著上升,3 h后表达量显著下降;caspase-4,5 酶活性在感染1~3 h内升高,3 h后降低。TLR4的mRNA及蛋白表达量均显著上调,CLI-095可抑制大肠杆菌对细胞的黏附作用。大肠杆菌感染成纤维细胞后LDH释放量增加、caspase-3 基因表达上调、caspase-4,5酶活性升高,促进了绵羊乳腺成纤维细胞的损伤、凋亡及焦亡;大肠杆菌感染促进绵羊乳腺成纤维细胞内TLR4 mRNA及蛋白的表达;TLR4阻断剂CLI-095可能通过抑制 TLR4的表达抑制E.coli对细胞的黏附作用。

关键词: 绵羊, 大肠杆菌, 乳腺炎, 成纤维细胞, TLR4

Abstract:

In order to study the damage of mammary fibroblasts in sheep E.coli mastitis and the mechanism of TLR4,primary sheep mammary fibroblasts was obtained through in vitro culture,and E.coli artificially infected cells to establish E.coli mastitis cell model.Analyze the effect of E.coli on cell injury and the role of TLR4 in E.coli mastitis.Enzymatic digestion combined with differential digestion to obtain primary sheep mammary fibroblasts;MTT method to detect cell viability and cytotoxicity of the TLR4 blocker CLI-095;LDH method detects the damage of E.coli to sheep mammary fibroblasts and determines the optimal MOI;qRT-PCR to detect the relative expression of caspase-3 and TLR4 mRNA and the blocking effect of CLI-095;colorimetric method to detect the enzymatic activity of pyroptosis protein caspase-4,5;WB to detect the relative expression of TLR4 protein;the plate viable bacteria count method was used to detect the effect of CLI-095 on the adhesion of E.coli to sheep mammary fibroblasts.Sheep mammary fibroblasts was isolated,and the results of MTT assay showed good cell viability.After sheep mammary fibroblasts were infected with E.coli MOI=4:1,the release of LDH was significantly increased in each time period;the relative expression of caspase-3 mRNA increased significantly within 1-3 h of infection,and decreased significantly after 3 h;caspase-4,5 enzyme activity increased within 1-3 h of infection,and decreased after 3 h.The mRNA and protein expression of TLR4 were significantly up-regulated,and the CLI-095 could inhibit the adhesion of E.coli to cells.After E.coli infects fibroblasts,the release of LDH was increased,the expression of caspase-3 gene was up-regulated,and the activity of caspase-4,5 was increased,which promotes the injury,apoptosis and pyroptosis of sheep mammary fibroblasts.E.coli infection promotes the expression of TLR4 mRNA and protein in sheep mammary fibroblasts.The TLR4 blocker CLI-095 may inhibit the adhesion of E.coli to cells by inhibiting the expression of TLR4.

Key words: Sheep, E.coli, Mastitis, Fibroblast, TLR4

马小军, 王加冕, 李世瑛, 颜丽娇, 麻强生, 张小丽. 大肠杆菌感染对绵羊乳腺成纤维细胞损伤及TLR4表达的影响[J]. 华北农学报, 2022, 37(2): 231-238. doi: 10.7668/hbnxb.20192742.

MA Xiaojun, WANG Jiamian, LI Shiying, YAN Lijiao, MA Qiangsheng, ZHANG Xiaoli. Effects of E.coli Infection on Fibroblast Injury and TLR4 Expression in Sheep Mammary Glands[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 231-238. doi: 10.7668/hbnxb.20192742.

http://www.hbnxb.net/CN/Y2022/V37/I2/231

图/表 14

表1 绵羊caspase-3引物信息

Tab.1 Sheep caspase-3 primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence	产物大小/bp Produce size	退火温度/℃ Tm	登录号 Accession
caspase-3 F	GGCTCTGAGTGTTTGGGGAA	131	60	XM-015104559.1
caspase-3 R	CCTGGACAAAGTTCCGTGGT

表2 绵羊TLR4引物信息

Tab.2 Sheep TLR4 primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence	产物大小/bp Produce size	退火温度/℃ Tm	登录号 Accession
TLR4 F	ACTGACGGGAAACCCTATCC	208	60	AY957615
TLR4 R	CAGGTTGGGAAGGTCAGAAA

图1 乳腺成纤维细胞形态

Fig.1 Mammary fibroblast morphology

图2 乳腺成纤维细胞Giemsa染色

Fig.2 Giemsa staining of mammary fibroblasts

图3 绵羊乳腺成纤维细胞生长曲线
不同小写字母表示差异显著(P<0.05);相同小写字母表示差异不显著(P>0.05)。图4,6—9,11同。

Fig.3 Growth curve of sheep mammary fibroblasts
Different lowercase letters show significant difference(P<0.05);The same lowercase letters indicate that the difference is not significant(P>0.05).The same as Fig.4,6—9,11.

图4 LDH释放量的变化

Fig.4 Changes in LDH release

图5 倒置显微镜观察大肠杆菌感染成纤维细胞

Fig.5 Inverted microscope observation of E.coli infected fibroblasts

图6 caspase-3 mRNA相对表达量

Fig.6 Relative expression of caspase-3 mRNA

图7 caspase-4,5酶活性变化

Fig.7 caspase-4,5 enzyme activity change

图8 CLI-095对细胞增殖率的影响

Fig.8 Effect of CLI-095 on cell proliferation rate

图9 TLR4 mRNA相对表达量

Fig.9 Relative expression of TLR4 mRNA

图10 TLR4蛋白表达

Fig.10 TLR4 protein expression

图11 TLR4蛋白相对表达量

Fig.11 Relative expression of TLR4 protein

表3 细菌黏附数及黏附阻断率

Tab.3 Bacterial adhesion number and adhesion blocking rate

组别 Group	黏附数/(cfu/mL) Adhesion number	黏附阻断率/% Adhesion blocking rate
TLR4非阻断组	40.3×10⁶ ± 2.3×10⁶	18.32
TLR4 non-blocking
TLR4阻断组	33.0×10⁶ ± 1.2×10⁶
TLR4 blocking

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