中国农业科学
中國農業科學
중국농업과학
SCIENTIA AGRICULTURA SINICA
2015年
8期
1624-1631
,共8页
史洪岩%贺綦%程敏%孙婴宁%李辉%王宁
史洪巖%賀綦%程敏%孫嬰寧%李輝%王寧
사홍암%하기%정민%손영저%리휘%왕저
鸡%HOPX%前脂肪细胞%增殖
鷄%HOPX%前脂肪細胞%增殖
계%HOPX%전지방세포%증식
chicken%HOPX%preadipocyte%proliferation
【目的】构建鸡HOPX基因（homeodomain only protein X）全长编码区（coding region sequence, CDS）的真核表达载体，转染鸡原代前脂肪细胞，探讨HOPX基因过表达对鸡原代前脂肪细胞增殖的影响。【方法】利用Primer Premier 5.0软件设计鸡HOPX基因CDS区上、下游引物，以AA肉鸡腹部脂肪组织的cDNA为模板，采用PCR扩增、克隆鸡HOPX基因全长CDS区，并将其亚克隆至真核表达载体（pCMV-HA vector），获得HOPX基因的真核表达载体pCMV-HA-HOPX。采用双酶切鉴定、测序及Western blotting方法分析鉴定pCMV-HA-HOPX。采用胶原酶法分离培养12日龄AA商品肉仔鸡腹部脂肪组织原代前脂肪细胞，瞬时转染pCMV-HA-HOPX，转染6 h时后消化细胞，按照每孔50000个细胞数接种12孔培养板，并在细胞贴壁0、24、48和72 h，分别采用显微镜观察和CCK-8细胞增殖检测试剂盒分析HOPX基因过表达对鸡原代前脂肪细胞增殖的影响；同时，利用TRIzol法提取组织和细胞总RNA，并反转录合成cDNA，采用Real-time RT-PCR方法分析细胞增殖标志基因CyclinD1和PCNA的mRNA表达。【结果】测序结果显示，鸡HOPX基因的全长CDS区大小为222 bp，与NCBI发布的鸡HOPX基因mRNA序列（NM_204556）一致；利用HA标签抗体的Western blotting分析显示，真核表达载体pCMV-HA-HOPX能够表达出预期大小的蛋白分子（约9.5kD），表明鸡HOPX基因的真核表达载体pCMV-HA-HOPX构建成功。显微镜观察发现，转染pCMV-HA-HOPX载体的细胞（HOPX过表达组）在细胞贴壁后培养24和48 h的细胞数量低于转染pCMV-HA vector空载体（空载体对照组）的细胞数量；CCK-8检测分析发现，转染pCMV-HA-HOPX载体细胞的吸光度值（OD值）在细胞贴壁后培养24、48和72 h都极显著低于空载体对照组（P＜0.01）。与细胞增殖检测结果相一致，细胞增殖标志基因表达检测分析发现，在细胞贴壁后培养24 h后，HOPX过表达组细胞Cyclin D1基因的mRNA表达量显著低于空载体对照组（P＜0.05）；在细胞贴壁后培养48 h时，HOPX过表达组的PCNA基因的mRNA表达量显著低于空载体对照组（P＜0.05）；在细胞贴壁后培养72 h时，HOPX过表达组的PCNA和Cyclin D1基因的表达量均极显著低于空载体对照组（P＜0.01）。【结论】HOPX基因过表达在体外抑制鸡原代前脂肪细胞的增殖。
【目的】構建鷄HOPX基因（homeodomain only protein X）全長編碼區（coding region sequence, CDS）的真覈錶達載體，轉染鷄原代前脂肪細胞，探討HOPX基因過錶達對鷄原代前脂肪細胞增殖的影響。【方法】利用Primer Premier 5.0軟件設計鷄HOPX基因CDS區上、下遊引物，以AA肉鷄腹部脂肪組織的cDNA為模闆，採用PCR擴增、剋隆鷄HOPX基因全長CDS區，併將其亞剋隆至真覈錶達載體（pCMV-HA vector），穫得HOPX基因的真覈錶達載體pCMV-HA-HOPX。採用雙酶切鑒定、測序及Western blotting方法分析鑒定pCMV-HA-HOPX。採用膠原酶法分離培養12日齡AA商品肉仔鷄腹部脂肪組織原代前脂肪細胞，瞬時轉染pCMV-HA-HOPX，轉染6 h時後消化細胞，按照每孔50000箇細胞數接種12孔培養闆，併在細胞貼壁0、24、48和72 h，分彆採用顯微鏡觀察和CCK-8細胞增殖檢測試劑盒分析HOPX基因過錶達對鷄原代前脂肪細胞增殖的影響；同時，利用TRIzol法提取組織和細胞總RNA，併反轉錄閤成cDNA，採用Real-time RT-PCR方法分析細胞增殖標誌基因CyclinD1和PCNA的mRNA錶達。【結果】測序結果顯示，鷄HOPX基因的全長CDS區大小為222 bp，與NCBI髮佈的鷄HOPX基因mRNA序列（NM_204556）一緻；利用HA標籤抗體的Western blotting分析顯示，真覈錶達載體pCMV-HA-HOPX能夠錶達齣預期大小的蛋白分子（約9.5kD），錶明鷄HOPX基因的真覈錶達載體pCMV-HA-HOPX構建成功。顯微鏡觀察髮現，轉染pCMV-HA-HOPX載體的細胞（HOPX過錶達組）在細胞貼壁後培養24和48 h的細胞數量低于轉染pCMV-HA vector空載體（空載體對照組）的細胞數量；CCK-8檢測分析髮現，轉染pCMV-HA-HOPX載體細胞的吸光度值（OD值）在細胞貼壁後培養24、48和72 h都極顯著低于空載體對照組（P＜0.01）。與細胞增殖檢測結果相一緻，細胞增殖標誌基因錶達檢測分析髮現，在細胞貼壁後培養24 h後，HOPX過錶達組細胞Cyclin D1基因的mRNA錶達量顯著低于空載體對照組（P＜0.05）；在細胞貼壁後培養48 h時，HOPX過錶達組的PCNA基因的mRNA錶達量顯著低于空載體對照組（P＜0.05）；在細胞貼壁後培養72 h時，HOPX過錶達組的PCNA和Cyclin D1基因的錶達量均極顯著低于空載體對照組（P＜0.01）。【結論】HOPX基因過錶達在體外抑製鷄原代前脂肪細胞的增殖。
【목적】구건계HOPX기인（homeodomain only protein X）전장편마구（coding region sequence, CDS）적진핵표체재체，전염계원대전지방세포，탐토HOPX기인과표체대계원대전지방세포증식적영향。【방법】이용Primer Premier 5.0연건설계계HOPX기인CDS구상、하유인물，이AA육계복부지방조직적cDNA위모판，채용PCR확증、극륭계HOPX기인전장CDS구，병장기아극륭지진핵표체재체（pCMV-HA vector），획득HOPX기인적진핵표체재체pCMV-HA-HOPX。채용쌍매절감정、측서급Western blotting방법분석감정pCMV-HA-HOPX。채용효원매법분리배양12일령AA상품육자계복부지방조직원대전지방세포，순시전염pCMV-HA-HOPX，전염6 h시후소화세포，안조매공50000개세포수접충12공배양판，병재세포첩벽0、24、48화72 h，분별채용현미경관찰화CCK-8세포증식검측시제합분석HOPX기인과표체대계원대전지방세포증식적영향；동시，이용TRIzol법제취조직화세포총RNA，병반전록합성cDNA，채용Real-time RT-PCR방법분석세포증식표지기인CyclinD1화PCNA적mRNA표체。【결과】측서결과현시，계HOPX기인적전장CDS구대소위222 bp，여NCBI발포적계HOPX기인mRNA서렬（NM_204556）일치；이용HA표첨항체적Western blotting분석현시，진핵표체재체pCMV-HA-HOPX능구표체출예기대소적단백분자（약9.5kD），표명계HOPX기인적진핵표체재체pCMV-HA-HOPX구건성공。현미경관찰발현，전염pCMV-HA-HOPX재체적세포（HOPX과표체조）재세포첩벽후배양24화48 h적세포수량저우전염pCMV-HA vector공재체（공재체대조조）적세포수량；CCK-8검측분석발현，전염pCMV-HA-HOPX재체세포적흡광도치（OD치）재세포첩벽후배양24、48화72 h도겁현저저우공재체대조조（P＜0.01）。여세포증식검측결과상일치，세포증식표지기인표체검측분석발현，재세포첩벽후배양24 h후，HOPX과표체조세포Cyclin D1기인적mRNA표체량현저저우공재체대조조（P＜0.05）；재세포첩벽후배양48 h시，HOPX과표체조적PCNA기인적mRNA표체량현저저우공재체대조조（P＜0.05）；재세포첩벽후배양72 h시，HOPX과표체조적PCNA화Cyclin D1기인적표체량균겁현저저우공재체대조조（P＜0.01）。【결론】HOPX기인과표체재체외억제계원대전지방세포적증식。
[Objective]The objective of this study was to construct the eukaryotic expression vector of chicken full-length HOPX gene and investigate the effect of HOPX gene overexpression on chicken preadipocytes proliferation.[Method]Using Primer Premier 5.0 software, a pair of primers was designed to amplify the full-length coding sequence (CDS) of chicken HOPX. The full-length coding sequence (CDS) of chicken HOPX gene was PCR amplified from the cDNA from the abdominal fat tissues of AA broiler chickens and cloned into pCMV-HA vector. Chicken preadipocytes were isolated from the abdominal fat tissues of 12-day-old AA broiler chicken by collagenase digestion, cultured and transfected with pCMV-HA-HOPX and pCMV-HA empty vector, respectively. Cell proliferation was assayed by microscopic examination and Cell Counting Kit-8 (CCK-8). Gene expression was measured by Western blotting and quantitative Real-time RT-PCR. [Result] The sequencing results showed that the full-length coding sequence of chicken HOPX gene is 222 bp and identical with NCBI reference sequence (NM_204556). Western blotting analysis showed that pCMV-HA-HOPX could correctly express the HA-tagged HOPX. The microscopic examination showed that the numbers of the preadipocytes transfected with pCMV-HA-HOPX were less than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h and 48 h after of cell adhesion. CCK-8 analysis showed that the OD values of the preadipocytes transfected with pCMV-HA-HOPX were significantly lower than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h, 48 h and 72 h after of cell adhesion (P<0.01). Consistently, at 24 h after of cell adhesion, the mRNA expression of CyclinD1 was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05);at 48 h after of cell adhesion, the mRNA expression of PCNA was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05);at 72 h after of cell adhension, the mRNA expression of CyclinD1 and PCNA were extremely significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.01)[Conclusion]HOPX gene overexpression in vitro inhibits chicken preadipocyte proliferation.
