Escherichia coli lipopolysaccharide modulates bovine luteal cell function.
Escherichia coli lipopolysaccharide modulates bovine luteal cell function.
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DOI：
10.1136/vr.161.20.695
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发表时间：
2007-11-17
期刊：
影响因子：
--
通讯作者：
Sheldon IM
中科院分区：
文献类型：
--
作者：
Grant E;Lilly ST;Herath S;Sheldon IM
牛生育力低下的一个重要原因是与产后子宫炎或子宫内膜炎相关的发情周期黄体期延长（Opsomer等，2000年）。这种子宫疾病通常与大肠杆菌的存在有关，并且在这些动物的子宫液和外周血浆中存在高浓度的细菌脂多糖（LPS） （Dohmen等人2000年，Mateus等人2003年，Williams等人2005年）。发情周期的持续时间由卵巢中黄体的存在决定。黄体是排卵后形成的临时内分泌腺，在黄体生成素（LH）的调节下分泌黄体酮，直到黄体溶解开始下一个卵泡期（Schams和Berisha， 2004）。子宫感染延长黄体寿命的潜在机制可能包括黄体溶解的破坏，这与LPS影响子宫内膜前列腺素分泌的变化有关（Herath等，2006）。另外，LPS可能对黄体细胞的功能有直接影响，因此本研究采用体外培养来验证这一范式。黄体是在屠宰场屠宰后立即从牛身上收集的。发情周期的阶段是根据Ireland和其他人（1980）的标准确定的，本研究只使用了黄体中期的黄体。黄体细胞分离和培养方法如前所述（Pate和Condon 1982）。简单地说，通过酶切黄体组织分离细胞，在添加了Dulbecco 's Modified Eagle 's培养基的营养混合物F-12 Ham （Sigma, Poole， UK）中，以2.5× 105个细胞/ml的密度洗涤并在24孔板中接种。36小时后更换培养基，处理细胞24至48小时，如结果所示。在治疗期结束时，收集上清液并在- 20℃下保存，直到用放射免疫法测量孕酮浓度，如前所述（Barret和Wathes 1990）。每孔细胞用生理盐水洗涤2次，应用胰蛋白酶（Accutase, Sigma），细胞悬浮在培养基中，用台盼蓝排除法和血细胞计计数活细胞数。在每个实验中，从至少3个黄体中进行三次培养，并在加湿培养箱中保持37℃，空气中CO2含量为5%。为了测试黄体细胞在体外是否具有功能，用LH处理黄体细胞（最终浓度分别为1、3、10和30 ng/ml； LH由Dr. Parlow， NIDDK， USA提供）；对照培养提供培养基。为了确定LPS的作用，我们将大肠杆菌O55: B5 LPS（0.1、0.3、1.0和3.0 μg/ml; Sigma, Poole， UK）单独施用，或在10 ng/ml LH或LPS拮抗剂多粘菌素B （1,2,4,8 μg/ml; Sigma, Poole， UK）存在的情况下施用。使用SPSS 14.0 （SPSS, Chicago， USA）中的一般线性模型对数据进行分析。数据以均数±SEM报告，P< 0.05为显著性。
An important cause of subfertility in cattle is prolonged luteal phases of the oestrous cycle associated with postpartum metritis or endometritis (Opsomer and others, 2000). This uterine disease is commonly associated with the presence of Escherichia coli and there are high concentrations of bacterial lipopolysaccharide (LPS) in the uterine fluid and peripheral plasma of these animals (Dohmen and others 2000, Mateus and others 2003, Williams and others 2005). The duration of the oestrous cycle is determined by the presence of a corpus luteum in the ovary. The corpus luteum is a temporary endocrine gland formed after ovulation that secretes progesterone under the regulation of luteinising hormone (LH) until luteolysis initiates the next follicular phase (Schams and Berisha, 2004). The mechanisms underlying the extended lifespan of the corpus luteum by uterine infection could include disruption of luteolysis associated with changes in endometrial secretion of prostaglandins by LPS (Herath and others, 2006). Alternatively, there may be a direct effect of LPS on the function of luteal cells, so the present study used in vitro culture to test this paradigm.Corpora lutea were collected from cattle immediately after slaughter at an abattoir. The stage of the oestrous cycle was determined using the criteria of Ireland and others (1980) and only corpora lutea from the mid-luteal phase were used in the present study. The method of luteal cell isolation and culture was as previously described (Pate and Condon 1982). Briefly, cells were isolated by enzymatic digestion of luteal tissue, washed and plated in 24 well plates at a density of 2.5× 105 cells/ml in supplemented Dulbecco’s Modified Eagle’s medium nutrient mixture F-12 Ham (Sigma, Poole, UK). The media was changed after 36 hours and treatments applied to the cells for 24 to 48 hours, as indicated in the results. At the end of the treatment period, the supernatants were collected and stored at− 20 C until progesterone concentrations were measured by radioimmunoassay, as previously described (Barret and Wathes 1990). The cells in each well were washed twice with saline, trypsin (Accutase, Sigma) was applied, the cells suspended in media, and the number of lives cells counted using the Trypan Blue exclusion method and a haemocytometer. For each experiment, cultures were conducted in triplicate from at least 3 corpora lutea and maintained at 37 C, 5% CO2 in air, in a humidified incubator. To test if the luteal cells were functional in vitro, they were treated with LH (at final concentrations of 1, 3, 10 and 30 ng/ml; LH courtesy of Dr. Parlow, NIDDK, USA); control cultures were supplied with media. To determine the effect of LPS, E. coli O55: B5 LPS (0.1, 0.3, 1.0 and 3.0 μg/ml; Sigma, Poole, UK) was applied alone, or in the presence of 10 ng/ml LH or the LPS antagonist, polymyxin B (1, 2, 4, 8 μg/ml; Sigma, Poole, UK). Data were analysed using a general linear model in SPSS ver 14.0 (SPSS, Chicago, USA). Data are reported as mean±SEM, and significance attributed when P< 0.05.
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影响因子：
1.7
作者：
Schams, D;Berisha, B
通讯作者：
Berisha, B
影响因子：
2.8
作者：
Opsomer, G;Gröhn, YT;de Kruif, A
通讯作者：
de Kruif, A
影响因子：
2.2
作者：
BARRETT, J;WATHES, DC
通讯作者：
WATHES, DC
影响因子：
2.8
作者：
Dohmen, MJW;Joop, K;Lohuis, JACM
通讯作者：
Lohuis, JACM
影响因子：
2.2
作者：
Mateus, L;da Costa, LL;Ziecik, AJ
通讯作者：
Ziecik, AJ