人毛乳头细胞中P16基因的检测及意义
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摘要
目的：毛乳头细胞（dermal papilla cells，DPCs）位于毛囊基底部，是构成毛乳头的主要间充质细胞，一簇特化的成纤维细胞。体内外具有诱导毛囊形成的能力成为毛乳头细胞显著的生物学特性。凝集性生长是毛乳头细胞最明显的特征。毛乳头细胞在毛囊的形态学发生及其周期性生长调控中处于中心环节，使其成为毛发疾病领域内研究的热点。细胞周期调控因子P16为CDK4的抑制蛋白，对细胞增殖周期起负调节过程，P16主要作用于细胞G1～S期的演进过程。已有研究证明，成纤维细胞进入衰老时P16基因过度表达，其持续表达导致了细胞衰老。高传代的毛乳头细胞逐渐失去凝集性生长特性，细胞增殖周期逐渐延长。由此推测可能毛乳头细胞失去凝集性生长特性可能与P16基因过度表达有关。本实验旨在通过检测分离培养的毛乳头细胞，应用实时荧光定量PCR法(reversetranscription real-time quantitative polymerase chain reaction，RT-QPCR)。从RNA水平检测P16基因表达水平，探讨毛P16基因表达水平与毛乳头细胞凝集性生长特性之间的关系，揭示体外培养的毛乳头细胞丧失凝集性生长特性的可能机制，为毛发疾病的临床治疗提供实验依据。
方法：
1人毛乳头细胞的分离培养及生长特性观察。采用改良毛乳头细胞培养法[1-2]，获得足够的细胞培养于15%的DMEM培养基中，置于37℃、湿度、5%的CO2培养箱内常规传代培养。倒置相差显微镜观察细胞及生长状态。
2α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)染色鉴定毛乳头细胞。
3应用实时荧光定量PCR法从转录水平检测不同传代次数人毛乳头细胞P16基因表达情况。同时设定阴性对照ddH2O（双蒸水），阳性对照人血白细胞，内参照为甘油醛-3磷酸脱氢酶（glyceraldehydes-3-phosphate dehydrogenase, GAPDH）。
4运用统计软件SPSS13.0对数据进行统计处理。
结果：
1在毛乳头细胞的分离过程中我们采用改良毛乳头细胞培养法，降低了劳动难度，成功进行了毛乳头细胞的分离培养。分离的毛乳头细胞贴壁迅速，具有凝集性生长的生长特性。此特性随着传代次数的增多逐渐减弱，一般6代以后消失。
2所获得的细胞经免疫组化染色胞浆内表达α-平滑肌肌动蛋白，α-SMA染色呈阳性。
3实时荧光定量PCR法从转录水平检测不同传代次数人毛乳头细胞P16基因表达情况。P16基因表达量虽然很微弱，但5代、6代、7代人毛乳头细胞P16基因表达量随着传代次数增加呈上升趋势。其中6代与5代相比，表达量有微弱的上升。7代与6代相比，表达量呈明显上升趋势。
结论：
1我们应用改良毛乳头细胞培养法，用较少的头皮标本成功分离到充足数量的毛乳头，且体外培养的毛乳头细胞贴壁迅速，α-SMA染色鉴定呈阳性，6代以前具有凝集性生长特性。
2P16基因在不同代次体外培养的人毛乳头细胞中表达，表达量随着传代次数的增加呈上升趋势。
3人毛乳头细胞呈凝集性生长状态时，P16基因的表达量随着细胞代次的增加呈轻微的上升趋势；人毛乳头细胞为非凝集性生长状态时，P16基因的表达量随着细胞代次的增加呈明显的上升趋势。
4P16基因可能在人毛乳头细胞由凝集性生长转化为非凝集性生长的过程中起了一定的作用。人毛乳头细胞由凝集性生长转化为非凝集性生长的过程可能与细胞衰老过程相关。
5实时荧光定量PCR是一种较为灵敏，精确的实验方法。
6P16基因将来有望成为治疗毛发疾病的崭新靶点。
Objective：Dermal papilla cells (DPCs) are the main mesenchymal cellswhich are located at the bottom of the hair follicle composing dermal papilla.DPCs are a cluster of specialized fibroblasts. The salient biologicalcharacteristics of DPCs are the ability to induce hair follicle formation in vivoand in vitro. The DPCs showed growth pattern of aggregation propertysignificantly. DPCs occur at the central link in the morphology of the hairfollicle and its cyclical growth regulation，so they are the research focuses inthe field of hair diseases.P16(cell cycle regulation factor)，inhibition ofCDK4，plays a negative adjustment process via blocking cells in G1~S phaseof the cell cycle. The study found that when human fibroblasts entersenescence, the P16gene was over-expressioned，sustained expression of P16led to the cell senescence. DPCs of late period gradually lose their aggregativegrowth characteristics, extended cell cycle. So we consider that P16gene mayplay an important role in the aggregative behavior of DPCs. Theoverexpression of P16gene may lead to gradual disappearing of thesubcultures. The objective of our experiment is to examine the expressionlevels of P16in DPCs at a transcriptional level by reverse transcriptionreal-time quantitative polymerase chain reaction(RT-QPCR)，from humanscalp and investigate the relationship between the expression of P16gene andthe aggregative behavior of DPCs, reveal the possible mechanisms of culturedhuman dermal papilla cells losing aggregative growth characteristics andprovide the experimental basis for the therapy for hair diseases.
Methods:
1Culture and Observe DPCs. Dermal papillas were isolated fromhuman scalp hair follicles by the method improved from two steps enzymemethod. DPCs were cultured in DMEM medium supplemented with15%heat inactivated fetal bovine serum and in a humidified incubator at37℃,95%air,5%CO2.The morphological changes of cells were detected by the reversemicroscopy.
2DPCs were identified by immunochemistry with α-SMA(α-smoothmuscle actin) antibody.
3We detected the expression of P16gene, and investigated theexpression of P16gene at a transcriptional level in different generations ofDPCs by RT-QPCR.Human white blood cells was used as a positivecontrol,ddH2O was regarded as a negative control, while GAPDH(glyceraldehyde-3-phosphate dehydrogenase) was the internal control.
4Statistic analyses: Data was analyzed by spss13.0for windows.
Results:
1The method for isolation of DPCs was improved from two stepsenzyme method, which not only reduced the work load the labor difficultybut also acquired cultured DPCs from human scalp successfully. The cellsshowed growth pattern of aggregation property and rapid adherence. Thecharacteristic of aggregative behavior could be phased out along with theircultivation. And after the sixth passage, the subcultures tend to fail tocoagulate usually.
2The expression of α-SMA of cultured DPCs by immunohist-ochemical method. α-SMA were expressed in the cytoplasm. The cells werepositive to smooth muscle actin alpha.
3We detected the expression of P16gene, and investigated theexpression of P16gene at a transcriptional level in different generations ofDPCs by reverse transcription real-time quantitative polymerase chainreaction(RT-QPCR). Although the expression level of P16gene of DPCs wasnot high，the expression level of P16increased with cell subculture. Theexpression level of P16gene of6thpassages was faintly higher than5thpassages，the former was significantly lower than the7thpassages.(P<0.05).
Conclusions:
1Abundant demal papillas were isolated from less specimens of adults' scalp with the methold which was improved from two stepsenzyme.The cells with rapid adherence were positive to smooth muscle actinalpha and were provided with typical aggregative behavior before the sixthpassage.
2P16were expressed in DPCs，and the expression level of p16increased with cell subculture.
3The expression level of P16in DPCs increased slightly with cellsubculture under aggregative condition, while increased apparently undernon-aggregative condition.
4P16gene may play a role in the process changing aggregativecondition into non-aggregative condition in cultured DPCs. The process ofDPCs may have a relation with cell senescence.
5Real-time PCR was a sensitive and accurate experimental methodestablished for detection of the lever of P16gene expression in laboratory.
6P16gene could be regarded as a potential target in therapeuticintervention for hair diseases in future.
方法：
1人毛乳头细胞的分离培养及生长特性观察。采用改良毛乳头细胞培养法[1-2]，获得足够的细胞培养于15%的DMEM培养基中，置于37℃、湿度、5%的CO2培养箱内常规传代培养。倒置相差显微镜观察细胞及生长状态。
2α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)染色鉴定毛乳头细胞。
3应用实时荧光定量PCR法从转录水平检测不同传代次数人毛乳头细胞P16基因表达情况。同时设定阴性对照ddH2O（双蒸水），阳性对照人血白细胞，内参照为甘油醛-3磷酸脱氢酶（glyceraldehydes-3-phosphate dehydrogenase, GAPDH）。
4运用统计软件SPSS13.0对数据进行统计处理。
结果：
1在毛乳头细胞的分离过程中我们采用改良毛乳头细胞培养法，降低了劳动难度，成功进行了毛乳头细胞的分离培养。分离的毛乳头细胞贴壁迅速，具有凝集性生长的生长特性。此特性随着传代次数的增多逐渐减弱，一般6代以后消失。
2所获得的细胞经免疫组化染色胞浆内表达α-平滑肌肌动蛋白，α-SMA染色呈阳性。
3实时荧光定量PCR法从转录水平检测不同传代次数人毛乳头细胞P16基因表达情况。P16基因表达量虽然很微弱，但5代、6代、7代人毛乳头细胞P16基因表达量随着传代次数增加呈上升趋势。其中6代与5代相比，表达量有微弱的上升。7代与6代相比，表达量呈明显上升趋势。
结论：
1我们应用改良毛乳头细胞培养法，用较少的头皮标本成功分离到充足数量的毛乳头，且体外培养的毛乳头细胞贴壁迅速，α-SMA染色鉴定呈阳性，6代以前具有凝集性生长特性。
2P16基因在不同代次体外培养的人毛乳头细胞中表达，表达量随着传代次数的增加呈上升趋势。
3人毛乳头细胞呈凝集性生长状态时，P16基因的表达量随着细胞代次的增加呈轻微的上升趋势；人毛乳头细胞为非凝集性生长状态时，P16基因的表达量随着细胞代次的增加呈明显的上升趋势。
4P16基因可能在人毛乳头细胞由凝集性生长转化为非凝集性生长的过程中起了一定的作用。人毛乳头细胞由凝集性生长转化为非凝集性生长的过程可能与细胞衰老过程相关。
5实时荧光定量PCR是一种较为灵敏，精确的实验方法。
6P16基因将来有望成为治疗毛发疾病的崭新靶点。
Objective：Dermal papilla cells (DPCs) are the main mesenchymal cellswhich are located at the bottom of the hair follicle composing dermal papilla.DPCs are a cluster of specialized fibroblasts. The salient biologicalcharacteristics of DPCs are the ability to induce hair follicle formation in vivoand in vitro. The DPCs showed growth pattern of aggregation propertysignificantly. DPCs occur at the central link in the morphology of the hairfollicle and its cyclical growth regulation，so they are the research focuses inthe field of hair diseases.P16(cell cycle regulation factor)，inhibition ofCDK4，plays a negative adjustment process via blocking cells in G1~S phaseof the cell cycle. The study found that when human fibroblasts entersenescence, the P16gene was over-expressioned，sustained expression of P16led to the cell senescence. DPCs of late period gradually lose their aggregativegrowth characteristics, extended cell cycle. So we consider that P16gene mayplay an important role in the aggregative behavior of DPCs. Theoverexpression of P16gene may lead to gradual disappearing of thesubcultures. The objective of our experiment is to examine the expressionlevels of P16in DPCs at a transcriptional level by reverse transcriptionreal-time quantitative polymerase chain reaction(RT-QPCR)，from humanscalp and investigate the relationship between the expression of P16gene andthe aggregative behavior of DPCs, reveal the possible mechanisms of culturedhuman dermal papilla cells losing aggregative growth characteristics andprovide the experimental basis for the therapy for hair diseases.
Methods:
1Culture and Observe DPCs. Dermal papillas were isolated fromhuman scalp hair follicles by the method improved from two steps enzymemethod. DPCs were cultured in DMEM medium supplemented with15%heat inactivated fetal bovine serum and in a humidified incubator at37℃,95%air,5%CO2.The morphological changes of cells were detected by the reversemicroscopy.
2DPCs were identified by immunochemistry with α-SMA(α-smoothmuscle actin) antibody.
3We detected the expression of P16gene, and investigated theexpression of P16gene at a transcriptional level in different generations ofDPCs by RT-QPCR.Human white blood cells was used as a positivecontrol,ddH2O was regarded as a negative control, while GAPDH(glyceraldehyde-3-phosphate dehydrogenase) was the internal control.
4Statistic analyses: Data was analyzed by spss13.0for windows.
Results:
1The method for isolation of DPCs was improved from two stepsenzyme method, which not only reduced the work load the labor difficultybut also acquired cultured DPCs from human scalp successfully. The cellsshowed growth pattern of aggregation property and rapid adherence. Thecharacteristic of aggregative behavior could be phased out along with theircultivation. And after the sixth passage, the subcultures tend to fail tocoagulate usually.
2The expression of α-SMA of cultured DPCs by immunohist-ochemical method. α-SMA were expressed in the cytoplasm. The cells werepositive to smooth muscle actin alpha.
3We detected the expression of P16gene, and investigated theexpression of P16gene at a transcriptional level in different generations ofDPCs by reverse transcription real-time quantitative polymerase chainreaction(RT-QPCR). Although the expression level of P16gene of DPCs wasnot high，the expression level of P16increased with cell subculture. Theexpression level of P16gene of6thpassages was faintly higher than5thpassages，the former was significantly lower than the7thpassages.(P<0.05).
Conclusions:
1Abundant demal papillas were isolated from less specimens of adults' scalp with the methold which was improved from two stepsenzyme.The cells with rapid adherence were positive to smooth muscle actinalpha and were provided with typical aggregative behavior before the sixthpassage.
2P16were expressed in DPCs，and the expression level of p16increased with cell subculture.
3The expression level of P16in DPCs increased slightly with cellsubculture under aggregative condition, while increased apparently undernon-aggregative condition.
4P16gene may play a role in the process changing aggregativecondition into non-aggregative condition in cultured DPCs. The process ofDPCs may have a relation with cell senescence.
5Real-time PCR was a sensitive and accurate experimental methodestablished for detection of the lever of P16gene expression in laboratory.
6P16gene could be regarded as a potential target in therapeuticintervention for hair diseases in future.
引文
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