| Sheep provide a large number of sheep products such as meat,skin,wool and cashmere for human beings,and occupy a pivotal position in animal husbandry production.Insulin-like growth factor I(IGF-?)gene is central to regulation and control of the growth and development in vertebrate species and it is also an important candidate gene to generate new strains of livestock,for which the most important targets are to improve production and quality of meat or wool.However,the IGF-? gene has an alternative splicing phenomenon during transcription,resulting in a variety of splicing isoforms with different biological functions.Among all the splicing isoforms of the IGF-? gene,the class I splice isoforms account for about 70% of the total amount of IGF-?,which plays a major role in regulating the growth and development of the body.The class I splice isoforms have two subtypes,Ea and Eb.The functional differences between the two splice isoforms remain unclear.In this study,IGF-? gene class I Ea and class I Eb splice isoforms were obtained by gene cloning,and the biological information analysis of the system was carried out,and their temporal and spatial expression changes were revealed.Subsequently,two types of I splice isoforms were constructed.The overexpression vector of the construct and the interfering Si RNA were transfected into sheep fetal skin and placenta-derived fibroblasts,respectively,up-regulating or inhibiting the expression levels of the two class I splice isoforms,and analyzing the two class I splice isoforms.The effects of fibroblast proliferation,activity and apoptosis were studied.The biological functions of two class I splice isoforms of IGF-1 gene were discussed.The results are as follows:(1)In this study,we successfully cloned the Ea-type and Eb-type full-length coding regions of the IGF-? gene class I splice isoforms.The IGF-? gene class I Ea type includes exons 1,3,4,and 6.The amplified target fragment is 534 bp in length and encodes 154 amino acids;the class I Eb type includes exons 1,3,4,and 5.The amplified fragment of interest is 610 bp long and encodes 188 amino acids.The two splicing isoforms are generally hydrophobic and hydrophilic,and are water-soluble amino acids.Both contain a signal peptide,which is a secreted protein,mostly located in the secretory pathway.The structure of the two is similar overall,and there are differences in the E domain.(2)The semi-quantitative RT-PCR method was used to detect the expression levels of two IGF-? gene class I splice isoforms in 12 tissues of fetal sheep,lamb and adult sheep.The results showed that IGF-? gene class I Ea type showed higher expression level in fetal sheep and lambs,and was significantly higher in heart,muscle,lung,kidney,intestine,stomach and skin than incorresponding tissues of mature sheep(P<0.01);the expression level of Eb type in fetal muscle,lung and skin tissues was higher than the adult sheep(P<0.05),and the expression level in the stomach and testis of lambs was higher than the adult sheep(P<0.05).In general,the expression levels of two class I splice isoforms of IGF-? gene in fetal sheep and lambs were higher than those in adult sheep,and the expression level of class I Ea was more abundant than that of class I Eb.(3)The whole fragments of the CDS region of two class I spliced IGF-? genes cloned were inserted into the overexpression vector pc DNA3.1-EGFP,and interference sites were designed to transfect the overexpression vector and Si RNA by liposomium-mediated method,with the transfection efficiency of about 40%(4)MTT assay was used to detect cell proliferation.The results showed that the proliferation of the Ea transfected cells was higher than the control group(P<0.01).The proliferation capacity of the Eb was higher than the control group(P<0.05).After inhibiting the expression levels of the two spliced isomers,the cell proliferation capacity was lower than the control group(P<0.01).Moreover,the effect of IGF-? gene on the proliferation of placental fibroblasts was stronger than that of skin fibroblasts compared with those of the two sources..(5)CCK8 method was used to detect the cell activity,and the results showed that: after class I Ea overexpression,the cell activity was extremely significantly higher than that of the control group(P<0.01),and after class I Eb overexpression,the cell activity was higher than the control group(P<0.05).When interferencing the expression level of class I Ea,the cell activity was lower than the control group(P<0.01),and after inhibiting the expression level of class I Eb,the cell activity was lower than the control group(P<0.05).In addition,the enhanced effect of IGF-? gene on the activity of placental fibroblasts was stronger than that of skin fibroblasts.(6)The cell cycle was detected by PI staining.The results showed that the ratio of cells in G1/G0 phase was significantly lower than the control group after class I Ea overexpression(P<0.01),and the S phase was significantly increased(P<0.01).After class I Eb overexpression,the percentage of cells in G1/G0 phase was also significantly decreased(P<0.05),and the S phase was significantly increased(P<0.05).After transfecting class I Ea interfering si RNA,the proportion of cells in G1/G0 phase was significantly increased(P<0.01),and the S phase was significantly decreased(P<0.01).The class I Eb interference group was compared with the control group.There was also a difference in cell ratio,but it did not reach a significant level of difference(P>0.05).Moreover,the IGF-? gene regulates the cycle in placental fibroblasts more strongly than skin fibroblasts.(7)Apoptosis was detected by Annexin V-PE/7-ADD double staining method.The results showed that the apoptosis rate of skin type and placental fibroblasts class I Ea and Eb overexpression groups were compared.The decrease was observed in the class I Ea overexpression group(P<0.01),and the apoptosis rate in the class I Eb overexpression group was significantly decreased(P<0.05).The apoptosis rate of the two groups of cells transfected with class I Ea interfering Si RNA was significantly increased(P<0.01).The apoptosis rate of group class I Eb interference group was not significantly different from that of the control group(P>0.05).And the inhibitory effect of IGF-? gene on apoptosis in placental fibroblasts is stronger than that of skin fibroblasts.. |
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