CN104498497A - miRNA related to milk goat mammary epithelial cell apoptosis and application thereof in breeding of milk goats - Google Patents

miRNA related to milk goat mammary epithelial cell apoptosis and application thereof in breeding of milk goats Download PDF

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CN104498497A
CN104498497A CN 201410757938 CN201410757938A CN104498497A CN 104498497 A CN104498497 A CN 104498497A CN 201410757938 CN201410757938 CN 201410757938 CN 201410757938 A CN201410757938 A CN 201410757938A CN 104498497 A CN104498497 A CN 104498497A
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milk
mammary epithelial
mir
cells
mirna
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CN 201410757938
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王建民
纪志宾
王桂芝
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山东农业大学
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Abstract

The invention relates to the technical field of genetic engineering, and provides miRNA related to milk goat mammary epithelial cell apoptosis and application thereof in breeding of milk goats. The small RNA molecule miR-143 is capable of inhibiting proliferation of mammary epithelial cells by delaying a cell cycle progress; by utilizing the characteristic, the inventor finds that miR-143 or precursor expression thereof can be inhibited through the genetic engineering technology; mammary epithelial cell apoptosis can be indirectly inhibited; therefore, the purposes of delaying mammary gland degradation, prolonging the lactation period and increasing the lactation amount of milk livestock can be achieved, and thus, the miRNA disclosed by the invention has high application value.

Description

一种与奶山羊乳腺上皮细胞凋亡相关的miRNA及其在奶山羊育种中的应用 And one kind of dairy goat mammary epithelial cell apoptosis related miRNA and its application in breeding dairy goats

技术领域 FIELD

[0001] 本发明涉及基因工程技术领域,提供了一种与奶山羊乳腺上皮细胞凋亡相关的miRNA及其在奶山羊育种中的应用。 [0001] The present invention relates to the field of genetic engineering, and to provide a dairy goat mammary epithelial cell apoptosis related miRNA and its application in breeding dairy goat.

背景技术 Background technique

[0002] 奶山羊乳腺发育、泌乳和退化过程具有一定规律性,其涉及乳腺细胞生长发育、增殖与凋亡,以及死亡。 [0002] Goat Mammary development, lactation and degradation processes have certain regularity, which relates to breast cell growth, proliferation and apoptosis, and death. 现在利用分子手段调控乳腺细胞凋亡过程、延缓乳腺退化过程,以延长奶山羊的泌乳期、提高泌乳量、改善奶山羊泌乳遗传性能,为崂山奶山羊的分子改良提供新的思路和有益的线索,是现今技术发展的主要方向。 Now the use of molecular means to control the process of apoptosis of breast, breast delaying the degradation process, in order to prolong lactation milk goat, improve milk yield and improve genetic performance of dairy goat lactation, provide new ideas and useful elements for the molecular improvement of Laoshan dairy goat , it is now the main direction of technology development.

[0003] miRNA是一类长度约22nt (nucleotide),存在于生物体内的、单链的、非蛋白质编码的、进化上高度保守的一种调控型的小RNA分子,其通过调控靶基因表达广泛参与了神经发育,细胞分化、增殖及凋亡,脂肪代谢(Xu et al,2003),乳腺发育等生理过程,并作为抑癌基因或癌基因而发挥重要的功能。 [0003] miRNA is a class of a length of about 22nt (nucleotide), present in the living body, small RNA molecules highly conserved, non-encoded protein, one regulatory evolution single stranded type, wide target gene regulated by involved in neural development, cell differentiation, proliferation and apoptosis, fat metabolism (Xu et al, 2003), mammary gland development and other physiological processes, and as oncogenes or tumor suppressor genes play an important function.

[0004] miRNA在动物中的研宄首先发现于线虫中,被称为异时开关基因的Lin-4,该基因虽然不编码蛋白但能使线虫发育事件延迟发生。 [0004] miRNA study based in animals was first discovered in nematodes, when the switch is referred to as iso Lin-4 gene, the gene encoding the protein, although not an event, but can delay occurs nematode development. 后来Reinhart等研宄人员又发现了第二个同样的异时开关基因Let-7,其能调控线虫发育时期的转变。 Later Reinhart et study based on a second person and found the same heterologous gene switch Let-7, which can be converted during the developmental regulation of nematodes. 后来研宄人员又发现Let-7 家族的很多miRNA,像miR-148、miR-84和miR-241也参与了线虫发育时期转变的调控。 Later, a Subsidiary officers also found a lot of miRNA Let-7 family, like miR-148, miR-84 and miR-241 is also involved in the regulation of nematode development period of change.

[0005] 随着测序技术的不断发展,特别是随着近几年来第二代测序技术的发展,研宄人员可以大批量地鉴定和发现一些新的miRNA,使miRNA的发展日新月益,鉴定出来的新miRNA成指数上升,miRNA以其独特的调控方式和表达特性迅速成为生物学领域研宄的热点。 [0005] With the development of sequencing technology, especially in recent years with the development of second-generation sequencing technology, the study based on large quantities can identify and discover new miRNA, the miRNA development of the rapidly growing, identified a new miRNA rise exponentially, miRNA regulation by its unique characteristics and expression quickly became the focus of study based on biology.

[0006] 乳腺发育、泌乳和退化过程中,激素、生长因子和一些蛋白质发挥着关键性作用。 [0006] mammary gland development, lactation and degradation process, hormones, growth factors, and some protein plays a key role. microRNA被发现后,它在器官发育过程中所发挥的作用也逐渐引起了研宄者的关注。 After microRNA was discovered, it is in the process of organ development in the role has gradually attracted the attention of a Subsidiary's. 2010年,来自哥廷根、法兰克福和汉诺威的科学家们发现激素和蛋白并非是乳腺发育的完全决定因素,一些小的核糖核酸分子在此过程中亦发挥了关键性作用,实验所用的小鼠拥有乳腺发育所必需的所有激素、生长因子和蛋白质,但由于缺乏miR-122和miR-132而导致小鼠乳腺导管完全无法发育,研宄人员第一次在动物模型中证实了小核糖核酸分子,即microRNAs,在器官发育中发挥重要的功能。 In 2010, scientists from Göttingen, Frankfurt and Hanover found hormones and proteins not entirely determinants of breast development, a number of small RNA molecules in this process has also played a key role in the mice used in the experiments has All breast development hormone necessary for protein and growth factors, but the lack of miR-122 and miR-132 in mice caused ductal not develop fully, for the first time confirmed the study based on the art of small RNA molecules in an animal model, That microRNAs, play an important function in organ development.

[0007] 而miR-143作为一种全新的小RNA分子,现有报道中记载其可以实现对于癌细胞的增殖抑制,进而可以用来抑制肿瘤的生长,但是由于癌细胞与正常细胞的差异,这一作用是否能够应用于正常细胞中还未可知,至今未见关于miR-143在乳腺细胞凋亡中的作用及其育种应用的任何报道。 [0007] and miR-143 as a new small RNA molecule, according to the prior reports which may be implemented for the inhibition of proliferation of cancer cells, and thus can be used to inhibit tumor growth, but the difference between normal cells and cancer cells due, whether this effect can be used in normal cells is still uncertain, so far no reports about any application and its breeding miR-143 in breast cell apoptosis of.

发明内容 SUMMARY

[0008] 本发明的发明人针对上述现有技术的情况,提供了一种与奶山羊乳腺上皮细胞凋亡相关的miRNA及其在奶山羊育种中的应用,该小RNA分子miR-143可以通过延缓细胞周期进程抑制乳腺上皮细胞的增殖,发明人利用该特性发现通过基因工程技术抑制miR-143 或其前体的表达,可间接抑制乳腺上皮细胞凋亡,以达到延缓乳腺退化、延长泌乳期、提高乳用家畜泌乳量,具有极高的应用价值。 [0008] The inventors of the present invention for the case of the above-described prior art, provides related miRNA and its application to the dairy goat mammary epithelial cell apoptosis in dairy goat breeding, the small RNA molecule by miR-143 delay cell cycle progression inhibit the proliferation of breast epithelial cells, the inventors found that by using the characteristic expression miR-143 or a precursor thereof is suppressed by genetic engineering techniques, can indirectly inhibit apoptosis in mammary epithelial cells, in order to achieve the breast retard degradation and prolong lactation to improve dairy cattle milk production, it has a very high value.

[0009] 发明人首先提供了一种与奶山羊乳腺上皮细胞凋亡相关的miRNA,该miRNA为miR-143,其基因序列如SEQ ID NO. 1所示,其前体基因序列如SEQ ID NO. 2所示。 [0009] The inventors first provided with a dairy goat mammary epithelial cell apoptosis related miRNA, the miRNA is miR-143, as its gene sequence SEQ ID NO. 1, the front gene sequences in SEQ ID NO . Figure 2.

[0010] 为了验证该miRNA的相关作用,发明人采用MTT法、荧光Hoechst/PI双染技术和流式细胞术研宄了miR-143对乳腺上皮细胞增殖与凋亡的影响,结果表明,miR-143通过延缓细胞周期进程,可抑制细胞增殖、促进凋亡;具体过程如下: [0010] In order to verify the effect of related miRNA, the inventors using the MTT assay, the fluorescent Hoechst / PI double staining and flow cytometry study based on the effect of miR-143 on proliferation and apoptosis in mammary epithelial cells, results showed that, miR -143 by delaying cell cycle progression, inhibit cell proliferation and promote apoptosis; process is as follows:

[0011] 发明人首先将miR-143瞬时转染到体外培养的原代乳腺上皮细胞,采用MTT法对细胞增殖结果进行检测,结果发现转染后48h和72h时,细胞活力在转染组比色值显著降低,在48h时两组间差异达到极显著水平(P〈0. 01),说明miR-143抑制乳腺上皮细胞的增殖; [0011] The inventors first miR-143 transiently transfected into cultured primary mammary epithelial cells, cell proliferation results detected by MTT assay, and found that at 48h and 72h after transfection, cell viability transfected ratio significantly lower color values, the difference between the two groups at 48h, was extremely significant (P <0 01.), described miR-143 inhibited the proliferation of mammary epithelial cells;

[0012] 在此工作的基础上,发明人进一步采用荧光Hoechst/PI荧光双染技术和流式细胞术验证了miR-143对乳腺上皮细胞凋亡的影响,并采用qRT-PCR检测了凋亡标志基因表达: [0012] Based on this work, the inventors further fluorescent Hoechst / PI double staining and flow cytometry to verify the effects of miR-143 on apoptosis in mammary epithelial cells, using qRT-PCR and detection of apoptosis marker gene expression:

[0013] 其荧光双染技术表明,在空白组Hoechst33342染色细胞核呈正常状态,对照组Hoechst33342也呈正常状态,而转染组经Hoechst33342染色后细胞核在荧光显微镜下则形态不完整,呈出碎裂状凋亡小体,表现出明显的凋亡现象; [0013] which show double staining technique, the nuclei were stained blank group Hoechst33342 normal control group also showed a normal state Hoechst33342, and then transfected by nuclear morphology under a fluorescence microscope after staining Hoechst33342 incomplete, the fragmentation was like apoptotic bodies, showed significant apoptosis;

[0014] 流式细胞仪分析发现,miR-143通过使细胞周期阻滞在Gl期,延缓了细胞周期的进行,对照组只检测到正常二倍体细胞DNA峰(Gl峰),转染组的Gl峰前出现一小峰,即所谓的凋亡峰(Ap峰),说明miR-143促进乳腺上皮细胞的凋亡; [0014] Flow cytometry analysis showed that, miR-143 by causing cell cycle arrest in Gl phase of the cell cycle delay in the control group only detected in normal diploid DNA peak (peak Gl), transfected a small peak in front of Gl peak, i.e., so-called apoptotic peak phase (Ap), indicating that miR-143 promotes apoptosis in mammary epithelial cells;

[0015] qRT-PCR检测结果表明,与对照组相比,凋亡基因BAX在转染组表达量极显著升高(P〈0. 01),BCL2在转染组其表达量变低,但差异不显著,进一步说明miR-143促进乳腺上皮细胞的凋亡。 [0015] qRT-PCR test results show that, compared with the control group, apoptotic gene BAX increased significantly (P <0. 01) the expression of transfected, the transfected group of BCL2 expression amount is low, but the difference not significant, explained further promote apoptosis miR-143 mammary epithelial cells.

[0016] 基于上述的发现,发明人进一步通过基因工程技术抑制miR-143在奶山羊体内的表达,可间接抑制乳腺上皮细胞凋亡,以达到延缓乳腺退化、延长泌乳期、提高乳用家畜泌乳量,具有极高的应用价值。 [0016] Based on the above findings, the inventors further suppress the expression of miR-143 in dairy goats, may indirectly inhibit apoptosis in mammary epithelial cells, in order to achieve the breast retard degradation and prolong lactation by genetic engineering techniques to improve lactation dairy cattle amount, with a high value. 除此之外还可以采用抑制基因序列如SEQ ID NO. 2所示的前体序列在奶山羊体内的表达,来实现上述的目的。 In addition to the precursor sequence as shown in inhibiting gene sequence SEQ ID NO. 2 expression in the milk of goats, to achieve the above objects may also be employed.

[0017] 综上所述,本发明的发明人在充分试验的基础上提供了一种与奶山羊乳腺上皮细胞凋亡相关的miRNA及其在奶山羊育种中的应用,该小RNA分子miR-143可以通过延缓细胞周期进程抑制乳腺上皮细胞的增殖,发明人利用该特性发现通过基因工程技术抑制miR-143或其前体在奶山羊体内的表达,可间接抑制乳腺上皮细胞凋亡,以达到延缓乳腺退化、延长泌乳期、提高乳用家畜泌乳量,具有极高的应用价值。 [0017] In summary, the present invention provides a dairy goat mammary epithelial cells and apoptosis related miRNA and its application in breeding dairy goats adequately tested on the basis of the small RNA molecule miR- 143 may delay cell cycle progression by inhibiting proliferation of mammary epithelial cells, the inventors found that by utilizing the characteristic genetic engineering techniques inhibiting miR-143 expression or a precursor thereof in dairy goats, may indirectly inhibit apoptosis mammary epithelial cells, in order to achieve breast delay degradation and prolong lactation, improve dairy cattle milk production, has a very high value.

附图说明 BRIEF DESCRIPTION

[0018] 图1为体外培养的原代乳腺上皮细胞免疫荧光鉴定结果灰度图, [0018] FIG. 1 is in vitro cultured primary mammary epithelial cells were identified by immunofluorescence results grayscale

[0019] 图中右下角深色区域是乳腺上皮细胞标志蛋白角蛋白18的免疫荧光鉴定;可见我们在体外培养获得的确实是乳腺上皮细胞; [0019] FIG dark areas in the lower right corner marker protein is a breast epithelial keratinocytes immunofluorescence 18; we obtain visible indeed in vitro mammary epithelial cell;

[0020] 图2为miR-143对细胞活力影响的MTT法检测结果示意图, [0020] FIG. 2 is a schematic diagram of the results of MTT assay miR-143 effects on cell viability,

[0021] 图中A为对照组与转染组细胞生长曲线;B为对照组与转染组MTT检测结果; [0021] A in the figure is a control and transfected cell growth curve; B is control and transfected MTT assay;

[0022] 图中A为通过细胞计数得到的对照组与转染组间细胞生长曲线,结果显示转染miR-143后,细胞计数明显减少,生长受到抑制;图中B为通过MTT法测得对照组与转染组间细胞吸光值结果,结果显示转染组细胞在培养48h时,吸光值明显降低;两部分结果都说明miR-143对体外培养的乳腺上皮细胞生长具有一定抑制作用; [0022] FIG. A is between control and transfected by cell counting obtained cell growth curves showed after transfection, miR-143, cell count decreased, growth was inhibited; figure B is obtained as measured by the MTT method between the control group and the transfected cells absorbance results showed transfected cells when cultured 48h, absorbance decreased; two partial results are described miR-143 grown with a certain inhibitory effect on cultured mammary epithelial cells;

[0023] 图3为miR-143对细胞凋亡影响的荧光Hoechst33342/PI双染检测结果灰度示意图, [0023] FIG. 3 is a miR-143 Effect on apoptosis fluorescence Hoechst33342 / PI double staining results gradation schematic,

[0024] 图中I 组(I -1,I -2,I -3)为阳性对照;II组(II -1,II -2, II -3)为转染组;III组(III -1,III -2, III -3)为阴性对照;1 列(I -1,II -1,III -1)为正常细胞;2 列(I -2,II -2, III -2)为PI 染色;3 列(I -3,II -3, III -3)为Hoechst33342 染色; [0024] FIG Group I (I -1, I -2, I -3) as positive control; group II (II -1, II -2, II -3) to transfection group; Group III (III -1 , III -2, III -3) negative control; 1 (I -1, II -1, III -1) normal cells; 2 (I -2, II -2, III -2) PI staining of ; 3 (I -3, II -3, III -3) is Hoechst33342 dye;

[0025] 经PI染色后,在显微镜下可观察到死亡细胞,经Hoechst33342染色后,可观察到凋亡细胞,并呈现凋亡颗粒;实验结果表明所有实验细胞经PI染色后,仅发现有少量细胞死亡,而大部分为正常生长细胞,而经H 〇echst33342染色后,在转染组呈现出明显的细胞凋亡状态,结果说明miR-143对体外培养的乳腺上皮细胞具有明显的促凋亡作用; [0025] Following PI staining, it can be observed under a microscope to cell death, after Hoechst33342 staining of apoptotic cells was observed, and the particles exhibit apoptosis; Experimental results show that all the cells were stained with PI, found only a small amount of cell death, whereas most normal cell growth, and stained with H 〇echst33342, showing a significant apoptotic state of the cells in the transfected cells, results demonstrate that miR-143 was capable of promoting apoptosis in mammary epithelial cells in vitro effect;

[0026] 图4为miR-143对细胞周期影响的流式细胞术检测结果示意图, [0026] FIG. 4 is a flow cytometry results of the influence of miR-143 on cell cycle schematic,

[0027] 图中A为阳性对照细胞周期分布;B为转染组细胞周期分布;C为不同组间细胞周期分布统计结果,显示经miR-143处理后,Gl期变长而S期变短,且两组处理间差异达到显著水平,结果说明miR-143可使体外培养的细胞生长周期阻滞在Gl期,从而延缓了细胞周期的进程; [0027] A in the figure is a positive control for cell cycle distribution; B is transfected cell cycle distribution; C distribution of statistical results of different cell cycle, show after miR-143 treatment, of Gl of variable length S phase becomes shorter and the difference between treatment groups was significant, the results can be described miR-143 in vitro cell cycle arrest in the Gl phase, thereby delaying the progression through the cell cycle;

[0028] 图5为miR-143对乳腺上皮细胞凋亡影响的流式细胞术检测结果示意图, [0028] FIG. 5 is a schematic flow cytometry results of miR-143 effects on breast epithelial cell apoptosis,

[0029] 图中A为阳性对照组;B为转染组, [0029] A in the figure is a positive control group; B is transfected group,

[0030] 结果显示在对照组仅检测到正常的二倍体细胞DNA峰(Gl峰),而转染组在Gl峰前出现一小峰,即所谓的凋亡峰(Ap峰).结果表明miR-143对体外培养的乳腺上皮细胞具有促凋亡作用; [0030] The results show only detected in the control group to normal diploid cell DNA peak (peak Gl), transfected group had a small peak in front of Gl peak, i.e., so-called apoptotic peak phase (Ap). The results show that miR -143 vitro mammary epithelial cells induce apoptosis;

[0031] 图6. miR-143过表达后凋亡相关基因BAX和BCL2表达的qRT-PCR检测结果示意图,结果显示凋亡基因BAX在转染组,与阳性对照相比其mRNA表达量显著升高,差异达到极显著(P〈0. 01),抗凋亡基因BCL2在对照组,与转染组相比其mRNA表达量显著升高,差异达到显著性(P〈〇. 05),结果表明miR-143对体外培养的乳腺细胞具有凋亡作用。 [0031] FIG. 6. miR-143 through apoptosis related gene BAX and qRT-PCR results showing the expression of BCL2 detected upon expression showed apoptotic gene BAX in transfection group, compared to the positive control of mRNA expression was significantly liters high, and the difference was significant (P <0. 01), the anti-apoptotic gene BCL2 in the control group, the mRNA expression was significantly increased as compared to transfected, the difference was significant (P <square. 05), the result miR-143 showed apoptotic effect on breast cells cultured in vitro.

具体实施方式 detailed description

[0032] 以下实施例中进一步定义本发明,根据以上的描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明精神和范围的情况下,可以对本发明作出各种改变和修改,以使其适用各种用途和条件。 [0032] The following examples further define the invention, the above description and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope of the invention, may be made to the present invention various changes and modifications to adapt it to various usages and conditions. 除特殊注明外,本发明所采用的均为本领域现有技术; Unless otherwise specified, are state of the art of the present invention is employed;

[0033] 其中细胞培养基的配制:体积比FBS !DMEM-F12为1:9的溶液,含有5mg/L牛胰岛素、5mg/L氢化可的松、10 μ g/L表皮生长因子和10万IU/L青链霉素双抗; [0033] wherein the cell culture medium formulation:! FBS DMEM-F12 volume ratio of 1: 9 solution containing 5mg / L bovine insulin, 5mg / L hydrocortisone, 10 μ g / L EGF and 100,000 IU / L penicillin-streptomycin double antibody;

[0034] 消化液为等体积混合的不含钙镁离子的PBS和0. 25wt%胰蛋白酶的EDTA溶液,消化条件为室温25°C,成纤维细胞消化时间为2-3分钟; [0034] The digestion solution was mixed in equal volumes of PBS free calcium and magnesium ions and 0. 25wt% trypsin EDTA solution protease digestion conditions at room temperature 25 ° C, fibroblasts digestion time is 2-3 minutes;

[0035] 其中所采用的FBS、DMEM-F12、0. 25wt%胰蛋白酶的EDTA溶液购自gibco公司, 牛胰岛素、氢化可的松和青链霉素双抗购自Sigma公司,表皮生长因子购自Invitrogen公司; [0035] FBS employed therein, DMEM-F12,0. EDTA solution 25wt% trypsin gibco available from the company, bovine insulin, hydrocortisone, and penicillin-streptomycin were purchased from Sigma double antibody, EGF available from Invitrogen Corporation;

[0036] 实施例1.原代乳腺上皮细胞培养 [0036] Example 1 Primary Mammary Epithelial Cell Culture

[0037] 1.乳腺组织采集与处理 [0037] 1. The breast tissue collection and processing

[0038] 用常规手段获取奶山羊乳腺组织,取样时,获取腺泡较丰富且结缔组织和脂肪较少的乳腺组织块约30g,放入含3%双抗的灭菌PBS中冲洗后,于低温2h内带回实验室。 After [0038] Goat Mammary tissue is acquired by conventional means, sampling, and less abundant than the acinar acquired connective tissue and breast adipose tissue mass of about 30g, placed in sterile PBS 3% bis-containing anti rinsed at back to the lab within 2h low temperature. 培养前将乳腺组织在培养皿中用含1 %双抗的PBS洗3-5次,直到组织颜色发白。 The preculture breast tissue culture dishes washed 3-5 times in PBS containing 1% by double antibody until a whitish color of the tissue. 用手术剪刀将组织剪碎(成Imm3左右的乳糜小块),再用含1 %双抗的PBS清洗2次,洗到组织块发白为止。 The minced tissue with surgical scissors (chyle into small pieces about Imm3), then with 1% bis antibody PBS twice, wash the white block until the tissue.

[0039] 2.组织块接种及培养 [0039] 2. The tissue pieces were inoculated and cultured

[0040] (1)用吸管或眼科镊子将乳糜样组织小块接种于培养瓶中:每个25cm2平皿十块左右均匀放置,每小块间距0. 5cm左右; [0040] (1) with a pipette or ophthalmic forceps chylous small tissue culture flasks were seeded to: about 25cm2 per ten evenly spaced plates, each small pitch of about 0. 5cm;

[0041] (2)将培养瓶轻轻翻转使组织块朝下,置于37°C培养箱中培养2_3h左右,使组织块牢固贴于培养瓶表面; [0041] (2) The tissue culture flask by gentle inversion block down on a cultured for 2_3h 37 ° C incubator, are firmly affixed to the tissue culture flask surface;

[0042] (3)加入含1 %双抗的全培养基3-4ml (培养基成分包括:DMEM/F12基础培养基、 10%胎牛血清FBS、胰岛素、氢化可的松和表皮生长因子),轻轻转动培养瓶,使培养液缓缓浸润组织块; [0042] (3) Add 3-4ml complete medium containing 1% of an anti-bis (medium composition comprising: DMEM / F12 basal medium, 10% fetal bovine serum FBS, insulin, hydrocortisone and epidermal growth factor) , gently turn flask, tissue culture solution gradually infiltrated mass;

[0043] (4)将培养瓶放入培养箱中于37°C、5% CO2条件下培养,每3天换液一次; [0043] (4) The flasks were placed in an incubator at 37 ° C, cultured under conditions of 5% CO2, medium was changed every 3 days;

[0044] (5) 10天后在显微镜下观察细胞爬出情况,待细胞长出后,每2天换液一次。 [0044] (5) after 10 days the cells were observed under a microscope to climb out, until the cells grow, medium was changed every 2 days.

[0045] 3.细胞纯化: [0045] 3. Cell Purification:

[0046] 在培养的乳腺上皮细胞中混有成纤维细胞,利用两种细胞消化和贴壁所需时间长短不同来进行纯化,待细胞生长良好后,用0. 25%的胰酶对细胞进行消化传代,最终获得较纯的乳腺上皮细胞,具体操作如下: [0046] After the cultured mammary epithelial cells mixed with fibroblasts, and digested using two cells adherent to a different length of time required for purification, until the cells grew well, with 0.25% trypsin the cells were digestion and passage, the finally obtained pure mammary epithelial cells, as follows:

[0047] (1)将培养瓶取出吸掉旧培养液,用卜2mLPBS润洗细胞,加入ImLO. 25%的胰酶消化; [0047] (1) The flasks were taken out of the old medium aspirated, the cells were washed with Bu 2mLPBS Run added ImLO 25% trypsin digestion.;

[0048] (2)放在显微镜下观察,成纤维细胞会比上皮细胞先从瓶壁上脱落,因此待有细胞脱落时弃掉消化液连同脱落的细胞,重新加入ImLO. 25%的胰酶继续消化,至细胞全部脱落时,加入全培养液终止消化; [0048] (2) was observed under a microscope, fibroblasts will start off on the sidewall than epithelial cells, and therefore have to be discarded when the cells were detached together with the digestive exfoliated cells, rejoin ImLO. 25% trypsin continues to digest, to a cell when all off, the whole broth was added to terminate the digestion;

[0049] (3)重复纯化3-4代,可消除成纤维细胞从而获得较纯的乳腺上皮细胞; [0049] (3) purification was repeated 3-4 generations, fibroblasts can be eliminated to obtain pure mammary epithelial cells;

[0050] 发明人对其进行体外培养的原代乳腺上皮细胞免疫荧光鉴定,结果如图1所示, 其中右下角深色区域是乳腺上皮细胞标志蛋白角蛋白18的免疫荧光鉴定;可见我们在在体外培养获得的确实是乳腺上皮细胞。 [0050] The inventors have its primary mammary epithelial cells were identified by immunofluorescence in vitro, the results shown in Figure 1, the lower right corner where dark areas are identified by immunofluorescence mammary epithelial cell marker cytokeratin 18 protein; in our visible in vitro indeed acquired mammary epithelial cells.

[0051] 实施例2.细胞瞬时转染 [0051] Example 2. Cells were transiently transfected

[0052] 转染试验分为3组:转染组、阴性对照和阳性对照 [0052] The transfection experiments were divided into three groups: transfection, negative control and positive control

[0053] 首先将miR-143按常规方法连接到pcDNA3. 1载体上获得转染质粒用于转染组; [0053] The miR-143 is first connected to a conventional manner to obtain a transfected plasmid pcDNA3 for transfection group on a carrier.;

[0054] 将pcDNA3. 1空载体作为阳性对照组的转染质粒; . [0054] The vector pcDNA3 1 empty plasmid as a transfection of the positive control group;

[0055] 阴性对照为不采用任何转染质粒; [0055] without using any negative control plasmid transfection;

[0056] 转染步骤按Lipofectamine™ LTX and PLUS™ Reagents (invitrogen)转染试剂盒说明书进行; [0056] Transfection procedures were carried out Lipofectamine ™ LTX and PLUS ™ Reagents (invitrogen) transfection kit instructions;

[0057] (1)对于培养贴壁良好处于指数生长期的细胞,用胰酶消化制成单细胞悬液,均匀等量的接种于24孔培养板; [0057] (1) good adherence to the culture in the exponential growth phase cells, digested with trypsin and made into single cell suspension, an equal amount of uniformly seeded in 24-well culture plates;

[0058] (2)培养24h后,细胞融合度达70-80 %时进行细胞转染; After [0058] (2) cultured 24h, cells reached confluence for cell transfection of 70-80%;

[0059] (3)将每组质粒及内参载体质粒pGL4. 74(转染质粒与内参质粒按质量比30:1)按每孔质粒终浓度〇· 8 μ g稀释至100 μ I Opti-MEM I中,质粒DNA( μ g)与Lipofectamine™ PLUS ( μ 1)体积按1:1的量加入Lipof ectamine™ PLUS后吹打混匀,5min后加入适量Lipofectamine™ LTX转染试剂轻轻吹打混匀,室温继续孵育30min ; [0059] (3) The internal reference vector plasmid and each plasmid pGL4 74 (with internal control plasmid transfected plasmid mass ratio 30: 1) the plasmid per well final concentration diluted 8-square · μ g to 100 μ I Opti-MEM I, the plasmid DNA (μ g) and Lipofectamine ™ PLUS (μ 1) by volume of 1: 1 was added after the amount of Lipof ectamine ™ PLUS mixed by pipetting, after adding an appropriate amount of Lipofectamine ™ LTX 5min transfection reagent mix gently pipetting, incubation was continued at room temperature for 30 min;

[0060] (4)加100 μ L混合液至待转染的每一细胞培养孔中,将含双抗的培养液(体积比FBS !DMEM-F12为1:9的溶液,含有5mg/L牛胰岛素、5mg/L氢化可的松、10 μ g/L表皮生长因子和10万IU/L青链霉素双抗;)换成不含抗生素的上述培养液,每孔加400 μ L,每组设置4个复孔; Culture wells per cell [0060] (4) was added 100 μ L mixture to be transfected, the culture medium containing the anti-bis (volume ratio FBS DMEM-F12 1:! 9 solution containing 5mg / L bovine insulin, 5mg / L hydrocortisone, 10 μ g / L EGF and 100,000 IU / L penicillin-streptomycin double antibody;) into the culture solution free of antibiotics, was added to each well 400 μ L, each group of 4 wells;

[0061] (5)将培养板放入37 °C 5% CO2培养箱中继续培养,在转染后6h换掉转染液,继续用正常培养液培养,转染后24-48h检测荧光强度。 [0061] (5) The plates were placed in 37 ° C 5% CO2 incubator and cultured, 6h post transfection replaced transfection solution, the culture was continued with normal culture, fluorescence intensity 24-48h after transfection .

[0062] 实施例3. MTT法检测miR-143对细胞增殖影响 [0062] Example 3. MTT assay miR-143 on cell proliferation

[0063] (1)细胞接种:用含10%胎牛血清的DMEM/F12培养液制成单细胞悬液,经过反复摸索,最终以每孔3 X IO4个细胞密度接种于96孔板,每孔培养液体积200 μ 1 ; [0063] (1) Cell Seeding: with DMEM containing 10% fetal bovine serum / F12 medium into single cell suspension, through trial and error, the final density of 3 X IO4 cells per well were seeded in 96-well plates, each pore volume of the culture solution 200 μ 1;

[0064] (2)细胞培养:将96孔培养板放入37 °C 5% CO2、饱和湿度的培养箱中培养; [0064] (2) Cell culture: The 96-well plates were placed in 37 ° C 5% CO2, humidified incubator;

[0065] (3)细胞转染:培养24h后,细胞生长到70-80%汇合度时,对每块96孔培养板同时进行转染,每板均分为3组(miR-143转染组,对照组(阳性),空白组(阴性)),每组设6个复孔,转染时用不含抗生素的培养基,转染后12h换成正常培养基; [0065] (3) cells transfected with: After 24h culture, when the cells were grown to 70-80% confluence on 96-well culture plate for each transfection at the same time, each plate was divided into three groups (miR-143 transfection group, control group (positive) and control group (negative)), each set of six parallel holes with medium without antibiotics, 12h after transfection the medium upon transfection into normal;

[0066] (4)细胞呈色:分别在转染的0h、48h、72h检测荧光值,每孔加入终浓度为5mg/ mlMTT溶液20 μ 1,继续孵育4h后终止培养。 [0066] (4) coloring the cells: transfected respectively 0h, 48h, 72h detecting fluorescence value added to each well to a final concentration of 5mg / mlMTT solution 20 μ 1, after the termination of culture incubation was continued 4h. 用注射器小心吸弃孔内全部上清液,同时每孔加入150 μ 1二甲基亚砜(DMSO),轻微振荡lOmin,使结晶物充分溶解; Carefully aspirate off with a syringe hole all the supernatant, and each well was added 150 μ 1 of dimethyl sulfoxide (DMSO), lOmin with gentle shaking, the crystal was fully dissolved;

[0067] (5)比色:混匀后用酶标仪在波长490nm处测定各孔吸光值,对获得的数据进行统计分析;通过比较过表达组与对照组的吸光值确定活细胞数(结果如图2所示)。 [0067] (5) color: After mixing each well was then determined by comparing the absorbance values ​​obtained data were statistically analyzed at 490nm wavelength; number of live cells is determined by comparing the absorbance value over expression group and the control group ( The results shown in FIG. 2).

[0068] 实施例4.荧光Hoechst33342/PI双染检测miR-143对细胞凋亡影响 EXAMPLE 4. Fluorescence Hoechst33342 / PI double staining miR-143 on apoptosis [0068] Embodiment

[0069] (1)固定培养的细胞消化后悬浮生长的细胞,lOOOrpm/min离心收集细胞,将IO5-IO6个细胞悬浮于ImL培养基中,再加入IOyL Hoechst 33342染液,混匀,37°C孵育5 〜15min ; [0069] (1) Cell cultures were fixed after digestion of cells grown in suspension, / min lOOOrpm cells were collected by centrifugation, the IO5-IO6 cells were suspended in ImL medium IOyL Hoechst 33342 dye was added, mixed, 37 ° C for 5 ~15min;

[0070] (2)细胞于4°C,500〜1000rpm/min离心5min弃去上清液; [0070] (2) Cell at 4 ° C, 500~1000rpm / min 5min centrifugation the supernatant was discarded;

[0071] (3)加入I. OiriLBuffei* A工作液(用双蒸水将lOXBuffei* A稀释10倍)悬浮细胞,加入5 μ L PI染液,室温避光放置5〜15min后混匀; [0071] (3) was added I. OiriLBuffei * A working solution (with double distilled water lOXBuffei * A 10-fold dilution) suspension cells, was added 5 μ L PI dye, dark place at room temperature after mixing 5~15min;

[0072] 对空白组,转染组和对照组均进行上述操作; [0072] for the control group, transfection and control groups are the above-described operation;

[0073] (4)荧光显微镜观察:Heochst 33342用氪激光激发的紫外光,将产生荧光(I -3, II -3, III-3),而凋亡细胞将表现为更为明亮(II -3) ;PI用氩离子激光激发荧光, 死亡细胞产生荧光(I -2, II -2,III-2);结果如图3所示。 [0073] (4) Fluorescence microscopy: Heochst 33342 krypton laser excitation with ultraviolet light, will emit fluorescence (I -3, II -3, III-3), and apoptotic cells will appear as a much brighter (II - 3); the PI fluorescence excited by argon ion laser, fluorescence dead cells (I -2, II -2, III-2); The results shown in Fig.

[0074] 实施例5.流式细胞术检测miR-143对细胞凋亡影响 Flow cytometry Example 5. Effect of miR-143 cells [0074] Embodiment

[0075] 1.细胞样品的制备: [0075] 1. Preparation of cell samples:

[0076] (1)用胰酶消化细胞,收集到5mL离心管中; [0076] (1) the cells were trypsinized, collected in 5mL centrifuge tube;

[0077] (2) 1000rpm/min 离心5min,弃掉上清液; [0077] (2) 1000rpm / min centrifuge 5min, the supernatant was discarded;

[0078] (3)加入预冷的PBS ImL并悬浮细胞,然后离心,弃掉并留少许上清液重要悬浮细胞; [0078] (3) was added and the suspension chilled PBS ImL cells, followed by centrifugation, the supernatant was discarded and leaving a little important cell suspension;

[0079] 2.细胞固定: [0079] 2. Cells were fixed:

[0080] 加入ImL冰浴预冷70%乙醇,轻轻吹打细胞以混匀,4°C过渡; [0080] The pre-cooling ice bath was added ImL 70% ethanol, mixing gently pipetting cells, 4 ° C transition;

[0081] 3. PI 染色: [0081] 3. PI staining:

[0082] (1)将固定好的细胞1000rpm/min离心5min,弃掉上清液; [0082] (1) A / min centrifugation good fixed cells 1000rpm 5min, the supernatant was discarded;

[0083] (2)加入冰浴预冷的PBS洗2次; [0083] (2) an ice bath was added washed twice with cold PBS;

[0084] (3)在离心管中加入PI染料并轻轻混匀,37°C避光温浴30min ; [0084] (3) was added PI dye tubes and mix gently, 37 ° C bath temperature for 30 min in the dark;

[0085] 4.流式检测与分析: [0085] 4. flow cytometry and analysis:

[0086] 用流式细胞仪在激发波长488nm波长处检测红色荧光,同时检测光散射情况。 [0086] detecting red fluorescence at a wavelength of 488nm excitation wavelength using a flow cytometer, light scatter occurs simultaneously detected. 采用适当分析软件进行细胞DNA含量分析和光散射分析,结果如图4和5所示。 Using appropriate analysis software cell DNA content analysis and light scattering analysis, the results shown in Figures 4 and 5.

[0087] 实施例6. qRT-PCR检测凋亡相关标志基因表达 Example 6. qRT-PCR marker of apoptosis-related gene [0087] Embodiment

[0088] (1)总RNA 提取及反转录:使用TIANGEN 公司的RNAsimple Total RNA Kit 总RNA 提取试剂盒(Cat. #DP419)提取细胞组织总RNA,具体操作参照试剂盒说明进行;将提取的RNA 米用TaKaRa 公司的RrimeScript™ RT reagent Kit (Cat. #RR037A)反转录合成cDNA 第一链,-20 °C保存备用。 [0088] (1) Total RNA extraction and reverse transcription: TIANGEN companies use RNAsimple Total RNA Kit Total RNA Extraction Kit (Cat # DP419.) Total RNA extraction tissue, with reference to the specific operation to kit instructions; extracted m using TaKaRa RNA's RrimeScript ™ RT reagent Kit (Cat. # RR037A) reverse transcription first strand cDNA synthesis, -20 ° C for use.

[0089] (2)引物设计说明:根据羊抗凋亡基因BCL2(NCBI登录号:JN036559. 1)和牛凋亡基因BAX(NCBI登录号:NM_173894. 1)序列设计引物,引物信息见如下表: [0089] (2) Primer design Description: See table below primers were designed, primer information according to the goat anti-apoptotic gene BCL2 (NCBI accession number:: JN036559 1.) And bovine apoptotic gene BAX (NM_173894 1 NCBI accession number):

[0090] [0090]

Figure CN104498497AD00081

[0091] (3) qRT-PCR 反应:以GAPDH 为内参基因,依照TaKaRa 公司的SYBR® Premix EX Taq™ II (DRR081A)说明书加样,反应体系为15μ1。 [0091] (3) qRT-PCR reactions: In GAPDH as reference gene, in accordance with instructions loaded TaKaRa company SYBR® Premix EX Taq ™ II (DRR081A), the reaction system was 15μ1.

[0092] (4)结果的分析统计:反应结果经ΜΧ3000Ρ配套软件对所得Ct值进行初步分析。 [0092] Statistical analysis (4) results: Results reaction ΜΧ3000Ρ supporting software resulting Ct values ​​were analyzed. 每个基因表达的相对值根据标准曲线运用T aact法进行计算,基因的表达水平表示为GT ± SE,用SAS9. 2软件中的one-way ANOVA法进行差异显著性分析,结果如图6所示凋亡基因BAX在转染组与对照组其相对表达水平分别为0. 9506和0. 6416, miR-143转染组BAX表达量极显著升高并达到极显著水平(P〈0. 01) ;BCL2在转染组与对照组其相对表达水平分别达到0. 4653和0. 5967,差异达到显著(P〈0. 05)),表明miR-143对体外培养的乳腺细胞具有凋亡作用。 Each gene expression relative value calculated based on a standard curve using T aact method represents gene expression level, a significant difference by analysis of GT ± SE SAS9. 2 software one-way ANOVA method, as shown in FIG 6 Results apoptotic gene BAX shown transfected with the control group relative expression levels thereof were 0.9506 and 0. 6416, miR-143 transfected BAX expression was significantly increased and reached a significant level (P <0. 01 ); the relative expression of BCL2 transfected with the control group reached the level of 0.4653 and 0.5967, the difference was significant (P <0 05)), showed that miR-143 in vitro apoptotic effect on breast cells. .

[0093] 试验例 [0093] Test Example

[0094] 发明人在获得了上述与奶山羊乳腺上皮细胞凋亡相关的,基因序列如SEQ ID NO. 1所示miRNA,以及基因序列如SEQ ID NO. 2所示其前体序列之后,利用常规基因工程技术抑制miR-143或其前体序列在奶山羊体内的表达,结果证实受试奶山羊的乳腺退化时间最短延缓了一周,最长延缓了三周,且泌乳量较未抑制的对照组平均提高8%,可见通过抑制miR-143或其前体的表达,可间接抑制乳腺上皮细胞凋亡,以达到延缓乳腺退化、延长泌乳期、提高乳用家畜泌乳量,具有极高的应用价值。 [0094] The inventors obtained with the above-described dairy goat mammary epithelial cell apoptosis related, such as the gene sequence SEQ ID NO. Shown miRNA, and gene sequence such as SEQ ID NO 1. 2, after which the precursor sequence shown by conventional genetic engineering techniques or inhibiting miR-143 precursor sequence in the expression dairy goats, the test results confirmed that the breast milk of goats week degradation time is the shortest delay, maximum delay of three weeks, and more lactation uninhibited control group, the average increase of 8%, showing that inhibition of expression by miR-143 or a precursor thereof, may indirectly inhibit apoptosis mammary epithelial cells, in order to achieve slow degradation of the breast, extended lactation, increase lactation dairy cattle, having a very high application value.

Claims (4)

  1. 1. 一种与奶山羊乳腺上皮细胞凋亡相关的miRNA,其特征在于:该miRNA为miR-143, 其基因序列如SEQ ID NO. 1所示。 A dairy goat mammary epithelial cells with apoptosis-related miRNA, wherein: the miRNA is miR-143, which gene sequence as shown in SEQ ID NO 1.
  2. 2. 根据权利要求1所述的miRNA,其特征在于:其前体基因序列如SEQ ID NO. 2所示。 2. miRNA according to claim 1, wherein: the front gene sequences as shown in SEQ ID NO 2.
  3. 3. 权利要求1所述的miRNA在奶山羊育种中的应用,其特征在于,抑制基因序列如SEQ ID NO. 1所示序列在奶山羊体内的表达。 Application of the miRNA in a dairy goat breeding in claim 1, characterized in that the gene sequence inhibits expression of the sequence as shown in SEQ ID NO. 1 in the milk of goats.
  4. 4. 根据权利要求3所述的应用,其特征在于,抑制基因序列如SEQ ID NO. 2所示的前体序列在奶山羊体内的表达。 4. The use according to claim 3, wherein the precursor sequence inhibits expression of the gene sequence as shown in SEQ ID NO. 2 in dairy goats.
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