梅花鹿鹿茸ColX基因的克隆、特性分析及有效siRNA筛选体系的建立
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摘要
为了探讨鹿茸的再生机制,本研究首先成功建立梅花鹿鹿茸软骨细胞体外短期培养方法,分析鹿茸软骨细胞体外培养特点。体外培养鹿茸干细胞,探讨了IGFⅠ对鹿茸干细胞的影响。根据牛、鼠、人、猪Collagen X(ColX)的保守序列设计引物,用分离培养的鹿茸肥大软骨细胞制作模板,通过RACE方法首次克隆、测定该目的基因,将基因登录GenBank获得的登录号为EF103187;同时将该基因序列构建到pEGFP-C1融合表达载体。根据目标基因构建针对性的短发夹RNA(shRNA)表达载体,用质粒表达的方式,获得了针对鹿茸软骨细胞ColX基因的小干扰RNA(siRNA),并将其共转染NIH-3T3细胞,检测siRNA对ColX的抑制效果,筛选有效siRNA,结果表明,不同组的shRNA较空载体对照组和无关对照组有效地抑制了ColX的表达。筛选得到对ColX具有高效抑制作用的shRNA重组表达质粒,为鹿茸软骨内成骨机理的研究奠定基础。
Deer antlers are the only mammalian appendage to display an annual cycle of full regeneration. The growth phase in antler involves the rapid proliferation of a number of tissues types including: epidermis, dermis, cartilage, bone and nerves. Antlers thus provide an excellent model to study the developmental regulation of these tissues. growth factors are implicated in pedicle formation and antler transformation in deer. However, the precise mechanism whereby growth factors control pedicle and antler formation is unknown. Markers of chondrogenesis used were type II collagen(ColII) and aggrecan, with type X collagen(ColX) being used as a marker of late-stage chondrocyte hypertrophy associated with endochondral ossification. However, there is some debate as to whether this process represents“true”endochondral ossification. As part of the characterization of the developmental process in pre-osseus antler tissue, Until recently, few transcriptional factors specifying its tissue-specific expression have been identified.
RNA interference (RNAi) refers to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell. This phenomenon can result in highly specific suppression of gene expression, which is mediated by 21 to 23 nucleotide small interfering RNA (siRNA) that is homologous in sequence to the silenced gene. RNAi can shut down or regulates gene expression in mammalian cells and confers as a cellular defense mechanism against gene invaders such as transposons and viruses. siRNA has showed its potential antiviral abilities in cultured mammalian cells and animals, and has been used as a new powerful tool for gene-specific therapeutics for viral disease and for gene function research..
Aim to explore the regeneration mechanism of the antler and establish the in vitro culture and expense methods of sika deer antler chondrocytes for the investigation of antler regeneration study and analyze the biological character of cultured antler chondrocytes. By the techniques of cell culture in vitro, the culture in vitro of the antler chondrocytes were performed. The growth of the cells was checked up by applying the ways of histology, MTT assay and immunohistochemistry. The results showed that the antler chondrocytes growth rapidly in plastic cell cultural board and chondrocytes l population demonstrates extensive proliferation and retains the capacity from original passage to passage 4. The original cells growth were faster than the passage. The antler chondrocytes were triangle, polygon and other shape. Immunochemistry results showd that the cultured cells were positive for ColII . Dyeing of toluidine blue was positive. The results suggested that the chondrocytes of isolated antler presented proliferous ability in culture by our method. Chondrocytes can be cultured and extensively proliferate and retain biological featurefor at least for 4 passage. The methods of antler chondrocytes culture can be used for the related continued research.
Using cell culture techniques to investigate the effcets of IGFⅠon the proliferation of antler stem cells from different growing period in vitro. Antler stem cells were isolated from sika deer antler of growing 30, 60, 90 day and cultured respectively. The results from the present study showed that the dependence of the antlerogenic cells on IGF1 for proliferation altered with the change of antler growth velocity. That is, the more faster the antler, the more stronger the antler stem cells stimulus response from IGFⅠin vitro. The most stimulus response of antler stem cells from IGFⅠin vitro was accord with the most quickly stage of antler growth velocity(60 day). Therefore we can see that the internal factors is the main factors determined the change of antler growth velocity.
ColX plays an important role in the enchondral bone formation. The methods of isolation and culture sika antler chondrocytes were established. Sika antler chondrocytes were cultured. Full-length cDNA of Sika ColX gene was amplified and cloned from total RNA of Sika antler chondrocytes by the RT-PCR and RACE methods(NO. EF103187). The cDNA sequence was 3 135 nt in length, including 96 nt of 5′untranslated region, 2 022 nt of coding region and 1 014 nt of 3′untranslated region. The coding region encoded a protein precursor with 674 amino acid residues. Amino acid identities of ColX between Sika and other vertebrates including cow, pig, dog, human and mouse were more than 82 %. Phylogenetic tree analysis based on the amino acid sequences showed that ColX of Sika and cow was in one clade.
Selection and validation of RNAi target sites. Aim to explore the function of the ColX gene in the regeneration of antler using small hairpin RNA (shRNA) expression vector. First as an assay for the siRNAs, we fused ColX gene to enhanced green fluorescent protein (EGFP) to provide a reporter system for monitoring siRNA function.
Construction and identification of pEGFP-ColX fusion gene plasmid. Full length cDNA fragment of ColX was obtained from amplified by Pyrobest DNA Polymerase. the fragment was inserted into a eukaryotic report vector pEGFP-C1. The fusion gene vector, named as pEGFP-ColX, was transformed into E. coli strain DH5αand identified by restricted endonuclease digestion. PCR and DNA sequences analysis. The results were shown as expected.The fluorescence of GFP in NIH-3T3 cells which was transfected with pEGFP-ColX plasmid DNA was detected successfully.
Construction and identification of different shRNA expression vectors. The small hairpin DNA (shDNA) sequences targeted to ColX gene for the siRNAs were determined by using the web-based criteria and were synthesized. Then the annealed shDNA was inserted into the pU6 vector. The positive clones were identified by DNA sequences analysis.
The changes of pEGFP-ColX images detected by FRCS and microscopy showed that different shRNA expression vectors were respectively transfected into NIH-3T3 cells with pEGFP-ColX. The fluorescence intensity of pEGFP-ColX in cells treated shRNA expression vectors trended to decrease and reached the lowest degree at 24～72h after transfection.The cDNA of ColX gene were derived from cells at 72h after transfection and were performed with real-time PCR. The expression of ColX gene mRNA of pU6-ColX-1, pU6-ColX-2, pU6-ColX-3 and pU6-ColX-4 groups reduced by 80.1%, 51.2%, 60.5% and 80.4% respectively compared to the control. The data of real-time PCR showed that mRNA tranxcription of ColX fusion gene was reduced by 80%compared to the empty vector control. The results suggested that the effectived sites of siRNA were found from ColX gene. They were be used to research the molecular modulation mechanism on endochondral ossification in the process of deer antler regeneration by interference the ColX with the siRNAs.
Deer antlers are the only mammalian appendage to display an annual cycle of full regeneration. The growth phase in antler involves the rapid proliferation of a number of tissues types including: epidermis, dermis, cartilage, bone and nerves. Antlers thus provide an excellent model to study the developmental regulation of these tissues. growth factors are implicated in pedicle formation and antler transformation in deer. However, the precise mechanism whereby growth factors control pedicle and antler formation is unknown. Markers of chondrogenesis used were type II collagen(ColII) and aggrecan, with type X collagen(ColX) being used as a marker of late-stage chondrocyte hypertrophy associated with endochondral ossification. However, there is some debate as to whether this process represents“true”endochondral ossification. As part of the characterization of the developmental process in pre-osseus antler tissue, Until recently, few transcriptional factors specifying its tissue-specific expression have been identified.
RNA interference (RNAi) refers to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell. This phenomenon can result in highly specific suppression of gene expression, which is mediated by 21 to 23 nucleotide small interfering RNA (siRNA) that is homologous in sequence to the silenced gene. RNAi can shut down or regulates gene expression in mammalian cells and confers as a cellular defense mechanism against gene invaders such as transposons and viruses. siRNA has showed its potential antiviral abilities in cultured mammalian cells and animals, and has been used as a new powerful tool for gene-specific therapeutics for viral disease and for gene function research..
Aim to explore the regeneration mechanism of the antler and establish the in vitro culture and expense methods of sika deer antler chondrocytes for the investigation of antler regeneration study and analyze the biological character of cultured antler chondrocytes. By the techniques of cell culture in vitro, the culture in vitro of the antler chondrocytes were performed. The growth of the cells was checked up by applying the ways of histology, MTT assay and immunohistochemistry. The results showed that the antler chondrocytes growth rapidly in plastic cell cultural board and chondrocytes l population demonstrates extensive proliferation and retains the capacity from original passage to passage 4. The original cells growth were faster than the passage. The antler chondrocytes were triangle, polygon and other shape. Immunochemistry results showd that the cultured cells were positive for ColII . Dyeing of toluidine blue was positive. The results suggested that the chondrocytes of isolated antler presented proliferous ability in culture by our method. Chondrocytes can be cultured and extensively proliferate and retain biological featurefor at least for 4 passage. The methods of antler chondrocytes culture can be used for the related continued research.
Using cell culture techniques to investigate the effcets of IGFⅠon the proliferation of antler stem cells from different growing period in vitro. Antler stem cells were isolated from sika deer antler of growing 30, 60, 90 day and cultured respectively. The results from the present study showed that the dependence of the antlerogenic cells on IGF1 for proliferation altered with the change of antler growth velocity. That is, the more faster the antler, the more stronger the antler stem cells stimulus response from IGFⅠin vitro. The most stimulus response of antler stem cells from IGFⅠin vitro was accord with the most quickly stage of antler growth velocity(60 day). Therefore we can see that the internal factors is the main factors determined the change of antler growth velocity.
ColX plays an important role in the enchondral bone formation. The methods of isolation and culture sika antler chondrocytes were established. Sika antler chondrocytes were cultured. Full-length cDNA of Sika ColX gene was amplified and cloned from total RNA of Sika antler chondrocytes by the RT-PCR and RACE methods(NO. EF103187). The cDNA sequence was 3 135 nt in length, including 96 nt of 5′untranslated region, 2 022 nt of coding region and 1 014 nt of 3′untranslated region. The coding region encoded a protein precursor with 674 amino acid residues. Amino acid identities of ColX between Sika and other vertebrates including cow, pig, dog, human and mouse were more than 82 %. Phylogenetic tree analysis based on the amino acid sequences showed that ColX of Sika and cow was in one clade.
Selection and validation of RNAi target sites. Aim to explore the function of the ColX gene in the regeneration of antler using small hairpin RNA (shRNA) expression vector. First as an assay for the siRNAs, we fused ColX gene to enhanced green fluorescent protein (EGFP) to provide a reporter system for monitoring siRNA function.
Construction and identification of pEGFP-ColX fusion gene plasmid. Full length cDNA fragment of ColX was obtained from amplified by Pyrobest DNA Polymerase. the fragment was inserted into a eukaryotic report vector pEGFP-C1. The fusion gene vector, named as pEGFP-ColX, was transformed into E. coli strain DH5αand identified by restricted endonuclease digestion. PCR and DNA sequences analysis. The results were shown as expected.The fluorescence of GFP in NIH-3T3 cells which was transfected with pEGFP-ColX plasmid DNA was detected successfully.
Construction and identification of different shRNA expression vectors. The small hairpin DNA (shDNA) sequences targeted to ColX gene for the siRNAs were determined by using the web-based criteria and were synthesized. Then the annealed shDNA was inserted into the pU6 vector. The positive clones were identified by DNA sequences analysis.
The changes of pEGFP-ColX images detected by FRCS and microscopy showed that different shRNA expression vectors were respectively transfected into NIH-3T3 cells with pEGFP-ColX. The fluorescence intensity of pEGFP-ColX in cells treated shRNA expression vectors trended to decrease and reached the lowest degree at 24～72h after transfection.The cDNA of ColX gene were derived from cells at 72h after transfection and were performed with real-time PCR. The expression of ColX gene mRNA of pU6-ColX-1, pU6-ColX-2, pU6-ColX-3 and pU6-ColX-4 groups reduced by 80.1%, 51.2%, 60.5% and 80.4% respectively compared to the control. The data of real-time PCR showed that mRNA tranxcription of ColX fusion gene was reduced by 80%compared to the empty vector control. The results suggested that the effectived sites of siRNA were found from ColX gene. They were be used to research the molecular modulation mechanism on endochondral ossification in the process of deer antler regeneration by interference the ColX with the siRNAs.
引文
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