| At present, along with the fast development of modern industry, the living environment of the human and wildlife is polluted in different degree by the Alkylphenol polyethoxylates (APEs) widely utilized in the production and life of the human and a great quantity phenolic discharged into environment. Nonylphenol (NP) with the stable chemical character, difficult to degrade, easy into organism, disturbing natural physiological activity, bring estrogen effect to reproductive and endocrine system of animals and lead decline of reproductive function, abnormal reproduction and disorder the well-balance of endocrine function in vertebrate. Steroidogenic acute regulatory protein (StAR) is a key factor of the steroidogenesis that make cholesterol carried from mitochondrial outer membrane into mitochondrial inner membrane and have follow-up response of the steroidogenesis continue. At present, there are abundant reports about APEs interfere pivotal enzyme of steroidogenesis in animals. Whereas, there were few investigations that NP has influencies amphibians StAR. This experiment takes Rana chensinensis as object, discuss the effects of NP to StAR in Leydig cells of R.chensinensis.The aim of this experiment is to reveal the mechanism of environmental chemical pollutants endanger the endocrine of amphibian, provide theoretical basis for decline of reproductive function in amphibian which caused by NP.It takes male R.chensinensis as experiment animals. Animals had been treated in 10-5,10-6,10-7M NP for 10d,20d,30d,0.1% ethanol as solvent comparison, then testis were got from R.chensinensis. Expressions of StAR mRNA are tested by in situ hybridizationand technique. The relative intensity of StAR are tested by immunohistochemical technique. The results and the conclusions are listed as follows:1. The value of RNA OD were mensured after total RNA's distillation, the ratio of OD260/ OD28 between 1.8 and 2.0, and appeared three bars which 28S,18S, 5S by electrophoretic examination, it shows, the purity of sample is eligible. We successfully expanded 273bp sequence of StAR cDNA, compare with StAR mRNA of Rana catesbeiana,Rana rugosa and Xenopus tropicalis the comparability were 96%,95%,75% respectively. We primarily confirm the sequence of that obtained is one fragment of StAR cDNA. Plasmid were distilled and sequenced after the fragment were transformed successfully. As a result, we ensure the insert fragment is aim fragment. At last, we synthesized RNA probe by linearization of plasmid.2. It used the probe that was synthesized before to carry through reaction of in situ hybridization in NP and control groups, the electropositive signals of StAR mRNA mostly expressed in Leydig cells, a small quantity of electropositive signals expressed in sertoli cell. It accordance with function of steroidogenesis in Leydig cells3. It checks out the StAR albumen of spermary by immunohistochemical method, the electropositive signals of StAR expressed in Leydig cells, a small quantity of electropositive signals expressed in sertoli cell. It accordance with expresive location of StAR hybridization in situ. The one way analysis of variance result with the relative intensity of StAR expression shows that in 10-5M NP group the comparison with the relative intensity and common of StAR expression in the treated times is a little low, the change has no significant difference (P>0.05).The 10-5M NP treatment can restrain the StAR expression, to infer this NP density has a lower grade toxicity. for animals and control the StAR expression. In 10-5M NP treated group StAR expression was enhanced along with treated times. In treated time, the comparison change of the relative intensity of StAR expression has significant (P<0.01).In 30d the expression of StAR is the largest and has additive effect with treated time. In 10-5M NP treated group StAR expression was enhanced along with treated time. In 10d treaed time the comparison with the relative intensity of StAR expression has no change, the change comparison in 20d and 30d is significant(P<0.01), whereas after 20d and 30d treated time the relative intensity of expression has basic alike. To infer this treated density, animals can weaken or clean disturbing effect by self-regulation. It shows that the StAR expression has dose effect relationship with NP.4. By the comparison in the same treated time in 10-5M NP group the relative intensity of StAR expression has lower than common time, in 10-7M NP group higher than common,and the change has no significant (P>0.05). In 10-6M NP group the relative intensity of StAR expression obviously has higher than common, and the change has great significant (P<0.01).In 20d treated group, the relative intensity of StAR expression in 10-5M NP treated group obviously has lower than common, and the change has significant (P<0.05). In 10-6M,10-7M NP treated group it has higher than common and the change has obviously significant (P<0.01).It shows that 10-6M,10-7M NP increase the expression of StAR. The relative intensity of StAR expression in 10-6M NP group is higher than its in 10-7M NP group. In 30d treated time the relative intensity of StAR expression has no significant difference (P>0.05) with common in 10-5M NP group, whereas in 10-6M NP,10-7M NP group it is higher than common with obviously significant (P<0.01). At the same time, the relative intensity of StAR expression in 10-6M NP group is even higher than its in 10-7M NP group. After 10-6M NP density group treated 30d, the relative intensity of StAR expression reaches the highest. It can infer that 10-6M is critical concentration that NP adjusts StAR. It shows that the relative intensity of StAR expression has obviously additive effect relationship with NP. 5. Ethanol as solvent comparison in this experiment, the expression of spermary StAR treated in water body of 0.1% ethanol don't show significant difference with blank. It shows that 0.1% ethanol hasn't an influence the expression of StAR.These results show:in a certain density scope, NP can effect the expression of key point enzymes StAR in Leydig cells and sustentacular cells of R.chensinensis. The high density NP can restrain the expression of StAR, the low density NP make the expression of StAR up-regulation. The expression of StAR controlled by NP can disturb secretory activity of animals. |
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