Method for regulating lactic acid secretion level of immature sertoli cells of pigs and application

A Sertoli cell, immature technology, applied in the field of regulating the lactic acid secretion level of immature porcine Sertoli cells, can solve the problems of complex structure and function, and achieve the level of simple method, increased lactate dehydrogenase activity, and lactic acid production by cells. reduced effect

Active Publication Date: 2022-03-08
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a comprehensive understanding of this chaperone remains challenging due to its inherent structural and functional complexity
[0004] Studies have found that in the testes of sexually mature pigs, spermat...

Method used

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  • Method for regulating lactic acid secretion level of immature sertoli cells of pigs and application
  • Method for regulating lactic acid secretion level of immature sertoli cells of pigs and application
  • Method for regulating lactic acid secretion level of immature sertoli cells of pigs and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction and transfection of HSP90AA1 overexpression vector

[0028] Referring to the HSP90AA1 sequence published on GenBank, a pair of primers were designed and synthesized to amplify the full length of HSP90AA1, the upstream primer 5′-GAGAACGCGTATGCCCGAGGAA-3′, and the downstream primer 5′-GAGAGCGGCCGCCTAATCGACTTC-3′. Using Sertoli cell cDNA as a template, use 2×PCR Solution Premix Prime The HS enzyme amplifies the HSP90AA1 gene fragment, and the estimated size of the amplified fragment is 2202bp. PCR reaction conditions: denaturation at 98°C for 10s, annealing at 60°C for 15s, extension at 72°C for 2min, 30 cycles. The results were observed by 1.0% agarose gel electrophoresis, and the accuracy was judged according to the size of the fragments. Cut the gel to recover the target fragment, measure the concentration, double enzyme digestion, connect the T vector overnight at 16°C and transform into competent Escherichia coli, pick positive clones for col...

Embodiment 2

[0030] Embodiment 2: RT-PCR, Western and cell immunofluorescence detection overexpression effect

[0031] RT-qPCR: The cells in the control group without transfection, the empty vector transfection group and the HSP90AA1 overexpression vector transfection group were collected, and the total RNA was extracted with Trizol, reverse-transcribed with ABI reverse transcription kit, Roche FastStart Universal STBR GreenMaster (Rox ) kit for qPCR, the PCR reaction conditions are as follows: 50°C for 2min, 95°C for 10min, 95°C for 15s, 60°C for 60s, 40 cycles, upstream primer: 5'-ATCGCCCAGTTGATGTCGTT-3', downstream primer: 5'-GTGAGGGTCCGGTCTTGCT- 3'. Using β-actin as an internal reference gene, the expression level was calculated using the 2-ΔΔCt method, and statistical analysis was performed using SPSS software.

[0032] Western: Collect the cells in the control group without transfection, the empty vector transfection group and the HSP90AA1 overexpression vector transfection group, l...

Embodiment 3

[0035] Example 3: Overexpression of HSP90AA1 can significantly increase the activity of lactate dehydrogenase and the production of lactic acid in pig immature Sertoli cells

[0036] Lactic acid determination: detected by a lactic acid detection kit. First, take 20 μL of the cell supernatant of the empty vector group and the overexpression group and add it to a 5 mL EP tube, add 1 mL of the working solution (1:100 from the enzyme stock solution and the enzyme dilution solution), and 200 μL of the chromogenic reagent into the EP tube. , mix well and place in a 37°C water bath for 10 minutes of reaction, then add 2 mL of stop solution to each tube), add the reacted samples to the microtiter plate, 200 μL / well, and repeat 3 times for each group. Absorbance detection was carried out under the condition of OD530nm of enzyme-linked immunosorbent detection instrument. Keeping all experimental conditions the same, three replicate experiments were performed. The levels of lactic acid...

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Abstract

The invention discloses a method for regulating the lactic acid secretion level of immature sertoli cells of pigs and application. The invention discloses a method for adjusting the lactic acid secretion level of a porcine immature sertoli cell line by changing the HSP90AA1 gene level, and belongs to the field of cell gene engineering. Specifically, the HSP90AA1 gene expression level is changed by transfecting an HSP90AA1 overexpression vector and a specific inhibitor to treat the immature testicular cells, and the lactic acid synthesis level of the immature testicular cells indicated by the HSP90AA1 level is judged by a method for determining the lactic acid generation and lactic dehydrogenase activity through enzyme-linked reaction. The result shows that the HSP90AA1 can positively regulate and control the lactic acid synthesis of the immature sertoli cells of the pigs, and can be used as a method for regulating the lactic acid secretion level of the immature sertoli cells of the pigs.

Description

technical field [0001] The invention belongs to the field of cell genetic engineering, and in particular relates to a method and application for regulating lactic acid secretion level of pig immature Sertoli cells. Background technique [0002] Leydig cells and Sertoli cells are the two most important types of somatic cells affecting spermatogenesis in animal testes. Without supporting cells, male germ cells cannot survive and mature in the body. Lactic acid produced by Sertoli cells can not only provide the necessary energy substrate for spermatogenesis, but also act as a paracrine factor to regulate the function of spermatogenic cells. It has also been shown that intratesticular infusion of lactic acid improves sperm production in adult cryptorchids. Therefore, regulating the level of lactate secretion in Sertoli cell lines of porcine immature testes is of great significance for the study of mammalian spermatogenesis. [0003] HSP90AA1 is a member of the HSP90s family a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N5/10C12N5/071C12P7/56
CPCC07K14/47C12N15/85C12N5/0683C12P7/56
Inventor 杨彩侠陈璐杜志强
Owner YANGTZE UNIVERSITY
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