应用mRNA差异显示技术筛选猪前脂肪细胞分化相关的基因
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摘要
脂肪组织的生长与脂肪细胞体积增大及前脂肪细胞分化成脂肪细胞有关。在前脂肪细胞分化过程中伴随着许多基因的特异性表达,细胞形态亦有相应的变化。猪是脂肪沉积能力很强的动物之一,脂肪在皮下组织和内脏器官的过度沉积是影响猪肉品质的重要因素。本研究采用mRNA差异显示技术筛选猪前脂肪细胞分化过程相关的差异表达基因,为进一步研究猪脂肪沉积代谢的调控机制提供科学依据。
试验1、猪前脂肪细胞的分离与鉴定
取3日龄健康仔猪颈部皮下脂肪组织,利用胶原酶消化法分离猪前脂肪细胞,进行原代培养,并采用MTT法、油红O染色、LPL活性测定来鉴定猪前脂肪细胞。结果显示,体外培养的猪前脂肪细胞在经历短暂的迟缓期后,进入对数生长期,直至第7天开始进入平台期,在培养第10天开始出现细胞调亡。细胞形态学观察发现,原代培养的猪前脂肪细胞从第3天开始充脂,细胞内脂肪滴被油红O染成红色,此后伴随细胞增殖和分化,充脂细胞逐渐增多,小脂滴不断汇聚形成大脂滴,细胞也由梭形逐渐变圆,体积增大。同时,LPL活性也呈现先上升后下降的变化趋势。上述结果证实猪前脂肪细胞已被成功分离。
试验2、猪前脂肪细胞分化过程差异表达基因的筛选
为进一步探讨猪前脂肪细胞分化过程中可能表达的调控基因,采用mRNA差异显示技术来筛选与其分化有关的差异表达基因。分别收集原代培养3天、5天、7天、10天的猪前脂肪细胞,用Trizol试剂提取细胞总RNA。通过RT-PCR和6%聚丙烯酰胺凝胶电泳,银染显色后,回收差异条带,并用PCR扩增,扩增片段克隆至pMD18-T载体中,测定其核苷酸序列,并将测序结果与Genbank中相关基因序列进行同源性比较。结果表明:
(1)采用DDRT-PCR技术成功获得20条EST序列,Blast在线分析结果表明,其中A22、A23、A33、A43、A55、A73、B42、C44等8个ESTs序列与Genbank数据库已报告的基因具有高度的同源性;A21、A32、A45、B41等4个ESTs序列与Genbank数据库已报告的基因同源性较低,其功能尚不明确;A31、A53、A24、B31、B71、C21、C62、C81等8个基因ESTs序列为本研究首次发现。
(2)A21、A23、A31、A32、A43、A53、B31、B71、C21、C62均表达于猪前脂肪细胞原代培养的第3天和第5天,可能与前脂肪细胞增殖有关;A22在前脂肪细胞培养的第3、5、7天表达,在第10天没有表达或者是表达量很低,推测A22的表达与前脂肪细胞增殖、分化的转换和调节有关;B41在培养第5、7天表达,可能与脂肪细胞的增殖有关;B42、C81在前脂肪细胞培养的第5、7、10天表达,可能与脂肪细胞的分化调节;A24、A33、A45、A73、C44均在猪前脂肪细胞原代培养的第7天和第10天表达,与脂肪细胞分化进程有关;A55与人的PURB基因同源性达到93%,尚不清楚A55是否参与脂肪细胞增殖或分化的调节。
Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from preadipocytes. During the preadipocyte differentiation process, is many specific genes are expressed and the cell morphology are changed accordingly. Porcine is one of the animal which has greatest fat deposit capacity, and the fat excessive accumulation in subcutaneous tissue and internal organs is an important factor which influences pork quality. In the present study, DDRT-PCR was used to screen genes associated with the porcine preadipocyte differentiation, to provide a scientific basis for the further study of regulation mechanism of fat metabolism in porcine.
Experiment 1. Subcutaneous Adipose Tissue from cervical part was removed from 3 d newborn crossbred piglets and Preadipocyte was obtained by collagenaseⅣDigestion. The isolated preadipocytes were cultured in 37℃, 5%CO2 and saturation humidity and subsequently identified by morphology observation, i.e, MTT or Oil Red O staining and detection of Lipoprotein Lipase( LPL ) activity. The results showed that the amount of preadipocytes tend to be exponentially increased. 3 days later, differentiating adipocytes filled with fatty droplet appeared. Henceforth, more adipocytes containing fatty droplet were observed, and the morphology of the cells had been changed from fibroblast-like to round. At the same time, the activities of lipoprotein lipase (LPL) tend to descend from higher level. Therefore, the cells came from subcutaneous adipose tissue digested by collagenaseⅣ, possess the typical characteristics of preadipocytes.
Experiment 2. In order to study the differential genes in which express on the process of proliferation and differentiation in the porcine preadipocytes cultured for 3, 5, 7 and 10 days were collected, and Trizol reagent was used to extract the total RNA of the cells. With RT-PCR and 6% non-polyacrylamide gel electrophoresis, the differential bands of the gene expression during the proliferation and differentiation period of the porcine preadipocytes were displayed on the silver stainined PAGE gel. Then the differential bands were sliced from the gel and the DNA segments were eluted and purified, the recovered products were subsequently used as template to amplify the fragments with PCR. Finanlly, the amplified segments were cloned into pMD18-T vector and sequenced, and the nucleotide sequence homology of the differential expressed gene and associated gene in Genbank were compared. The results showed that:
(1) 20 ESTs of the differential expressed gene were identified, and the result of blast analysis on line indicated that 8 ESTs, A22, A23, A33, A43, A55, A73, B42 and C44 shared highly nucleotide sequence homology with associated genes reported in Genbank, 4 ESTs, A21, A32, A45 and B41 shared relatively lower nucleotide sequence homology, and their founction remained unclear, and it was found for the first time that 8 ESTs, A31, A53, A24, B31, B71, C21, C62 and C81, which were not reported in Genbank, were associated with proliferation and differentiation of the cultured porcine preadipocytes in vitro.
(2) 10 ESTs, A21, A23, A31, A32, A43, A53, B31, B71, C21 and C62 mainly expressed in the pocine preadipocytes culured for 3 and 5 days, implied that genes had ESTs above were associated with preadipocyte proliferation; A22 expressed in the pocine preadipocytes culured for 3, 5 and 7 days, and the expression level was very low to be detected in cells cultured for 10 days, and presumed that A22 expression was related to the conversion and regulation process of proliferation and differentiation of the porcine preadipocytes in vitro. B41 expressed in the pocine preadipocytes culured for 5 and 7 days, implied that genes had ESTs above were associated with preadipocyte differentiation; B42, C81 expressed in the pocine preadipocytes culured for 5, 7 and 10 days,implied that genes had ESTs above were associated with preadipocyte proliferation; Furthmore, A24, A33, A45, A73 and C44 expression were found in adipocytes cultured for 7 and 10 days, which were relevant to preadipocyte differentiation. The nucleotide sequence homology of A55 and PURB originated from human came to 93%, and it as not clear that A55 was involved in regulatiuon of proliferation and differentiation of the porcine preadipocytes.
试验1、猪前脂肪细胞的分离与鉴定
取3日龄健康仔猪颈部皮下脂肪组织,利用胶原酶消化法分离猪前脂肪细胞,进行原代培养,并采用MTT法、油红O染色、LPL活性测定来鉴定猪前脂肪细胞。结果显示,体外培养的猪前脂肪细胞在经历短暂的迟缓期后,进入对数生长期,直至第7天开始进入平台期,在培养第10天开始出现细胞调亡。细胞形态学观察发现,原代培养的猪前脂肪细胞从第3天开始充脂,细胞内脂肪滴被油红O染成红色,此后伴随细胞增殖和分化,充脂细胞逐渐增多,小脂滴不断汇聚形成大脂滴,细胞也由梭形逐渐变圆,体积增大。同时,LPL活性也呈现先上升后下降的变化趋势。上述结果证实猪前脂肪细胞已被成功分离。
试验2、猪前脂肪细胞分化过程差异表达基因的筛选
为进一步探讨猪前脂肪细胞分化过程中可能表达的调控基因,采用mRNA差异显示技术来筛选与其分化有关的差异表达基因。分别收集原代培养3天、5天、7天、10天的猪前脂肪细胞,用Trizol试剂提取细胞总RNA。通过RT-PCR和6%聚丙烯酰胺凝胶电泳,银染显色后,回收差异条带,并用PCR扩增,扩增片段克隆至pMD18-T载体中,测定其核苷酸序列,并将测序结果与Genbank中相关基因序列进行同源性比较。结果表明:
(1)采用DDRT-PCR技术成功获得20条EST序列,Blast在线分析结果表明,其中A22、A23、A33、A43、A55、A73、B42、C44等8个ESTs序列与Genbank数据库已报告的基因具有高度的同源性;A21、A32、A45、B41等4个ESTs序列与Genbank数据库已报告的基因同源性较低,其功能尚不明确;A31、A53、A24、B31、B71、C21、C62、C81等8个基因ESTs序列为本研究首次发现。
(2)A21、A23、A31、A32、A43、A53、B31、B71、C21、C62均表达于猪前脂肪细胞原代培养的第3天和第5天,可能与前脂肪细胞增殖有关;A22在前脂肪细胞培养的第3、5、7天表达,在第10天没有表达或者是表达量很低,推测A22的表达与前脂肪细胞增殖、分化的转换和调节有关;B41在培养第5、7天表达,可能与脂肪细胞的增殖有关;B42、C81在前脂肪细胞培养的第5、7、10天表达,可能与脂肪细胞的分化调节;A24、A33、A45、A73、C44均在猪前脂肪细胞原代培养的第7天和第10天表达,与脂肪细胞分化进程有关;A55与人的PURB基因同源性达到93%,尚不清楚A55是否参与脂肪细胞增殖或分化的调节。
Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from preadipocytes. During the preadipocyte differentiation process, is many specific genes are expressed and the cell morphology are changed accordingly. Porcine is one of the animal which has greatest fat deposit capacity, and the fat excessive accumulation in subcutaneous tissue and internal organs is an important factor which influences pork quality. In the present study, DDRT-PCR was used to screen genes associated with the porcine preadipocyte differentiation, to provide a scientific basis for the further study of regulation mechanism of fat metabolism in porcine.
Experiment 1. Subcutaneous Adipose Tissue from cervical part was removed from 3 d newborn crossbred piglets and Preadipocyte was obtained by collagenaseⅣDigestion. The isolated preadipocytes were cultured in 37℃, 5%CO2 and saturation humidity and subsequently identified by morphology observation, i.e, MTT or Oil Red O staining and detection of Lipoprotein Lipase( LPL ) activity. The results showed that the amount of preadipocytes tend to be exponentially increased. 3 days later, differentiating adipocytes filled with fatty droplet appeared. Henceforth, more adipocytes containing fatty droplet were observed, and the morphology of the cells had been changed from fibroblast-like to round. At the same time, the activities of lipoprotein lipase (LPL) tend to descend from higher level. Therefore, the cells came from subcutaneous adipose tissue digested by collagenaseⅣ, possess the typical characteristics of preadipocytes.
Experiment 2. In order to study the differential genes in which express on the process of proliferation and differentiation in the porcine preadipocytes cultured for 3, 5, 7 and 10 days were collected, and Trizol reagent was used to extract the total RNA of the cells. With RT-PCR and 6% non-polyacrylamide gel electrophoresis, the differential bands of the gene expression during the proliferation and differentiation period of the porcine preadipocytes were displayed on the silver stainined PAGE gel. Then the differential bands were sliced from the gel and the DNA segments were eluted and purified, the recovered products were subsequently used as template to amplify the fragments with PCR. Finanlly, the amplified segments were cloned into pMD18-T vector and sequenced, and the nucleotide sequence homology of the differential expressed gene and associated gene in Genbank were compared. The results showed that:
(1) 20 ESTs of the differential expressed gene were identified, and the result of blast analysis on line indicated that 8 ESTs, A22, A23, A33, A43, A55, A73, B42 and C44 shared highly nucleotide sequence homology with associated genes reported in Genbank, 4 ESTs, A21, A32, A45 and B41 shared relatively lower nucleotide sequence homology, and their founction remained unclear, and it was found for the first time that 8 ESTs, A31, A53, A24, B31, B71, C21, C62 and C81, which were not reported in Genbank, were associated with proliferation and differentiation of the cultured porcine preadipocytes in vitro.
(2) 10 ESTs, A21, A23, A31, A32, A43, A53, B31, B71, C21 and C62 mainly expressed in the pocine preadipocytes culured for 3 and 5 days, implied that genes had ESTs above were associated with preadipocyte proliferation; A22 expressed in the pocine preadipocytes culured for 3, 5 and 7 days, and the expression level was very low to be detected in cells cultured for 10 days, and presumed that A22 expression was related to the conversion and regulation process of proliferation and differentiation of the porcine preadipocytes in vitro. B41 expressed in the pocine preadipocytes culured for 5 and 7 days, implied that genes had ESTs above were associated with preadipocyte differentiation; B42, C81 expressed in the pocine preadipocytes culured for 5, 7 and 10 days,implied that genes had ESTs above were associated with preadipocyte proliferation; Furthmore, A24, A33, A45, A73 and C44 expression were found in adipocytes cultured for 7 and 10 days, which were relevant to preadipocyte differentiation. The nucleotide sequence homology of A55 and PURB originated from human came to 93%, and it as not clear that A55 was involved in regulatiuon of proliferation and differentiation of the porcine preadipocytes.
引文
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