CN114150017B - Method for regulating lactic acid secretion level of pig immature testis support cells and application - Google Patents

Abstract

A method for regulating lactic acid secretion level of pig immature testis support cell and its application are provided. The invention discloses a method for regulating the lactic acid secretion level of a pig immature testis support cell line by changing the HSP90AA1 gene level, belonging to the field of cell genetic engineering. Specifically, the HSP90AA1 overexpression vector and the specific inhibitor are transfected to treat the pig immature testis support cells to change the expression level of the HSP90AA1 gene, and the method for measuring the lactic acid generation and the lactic acid dehydrogenase activity through enzyme-linked reaction is used for judging the lactic acid synthesis level of the pig immature testis support cells, so that the result shows that the HSP90AA1 can positively regulate and control the lactic acid synthesis of the pig immature testis support cells and can be used as a method for regulating the lactic acid secretion level of the pig immature testis support cells.

Description

Method for regulating lactic acid secretion level of pig immature testis support cells and application

Technical Field

The invention belongs to the field of cell genetic engineering, and particularly relates to a method for regulating the lactic acid secretion level of a pig immature testis support cell and application thereof.

Background

Mesenchymal cells and supporting cells are the two most important types of somatic cells in animal testes that affect spermatogenesis. Without support cells, male germ cells are not viable and mature in vivo. Lactic acid produced by the support cells can provide the necessary energy substrate for spermatogenesis and can also act as a paracrine factor to regulate the function of spermatogenic cells. Also, it has been shown that intratesticular delivery of lactic acid can enhance the production of adult cryptorchid sperm. Thus, modulation of lactate secretion levels in porcine immature testis support cell lines is of great interest in studying mammalian spermatogenesis.

HSP90AA1 is one of HSP90s family members, is an ATP-dependent molecular chaperone, and utilizes its atpase activity to drive conformational changes of the cofactor, promote folding, stabilization and activation of the protein of interest, and affect a variety of cellular functions. However, there remains a challenge to fully understand this chaperone protein due to its inherent structural and functional complexity.

Studies have found that in sexually mature pig testes, spermatogonia, spermatocyte, round sperm and elongated sperm cells express Hsp90, but no application of HSP90AA1 in regulating lactic acid secretion levels of pig immature testis supporting cells has been reported yet.

In order to change the expression level of HSP90AA1 in a pig testis support cell, the invention adopts the transfection of HSP90AA1 over expression and HSP90 specific inhibitor 17-AAG treatment in the pig testis support cell to increase or decrease the expression level of HSP90AA1, and then adopts an ELISA method to detect the regulation of the expression of the HSP90AA1 on the lactic acid generation level of a pig immature testis support cell.

Disclosure of Invention

The invention aims at: (1) Providing an application of improving lactic acid secretion of a swine immature testis support cell by over-expressing HSP90AA 1; (2) The use of reducing expression of HSP90AA1 in porcine immature testis support cells by 17-AAG treatment to reduce lactate secretion is provided.

In order to achieve the above object, the present invention is realized by the following technical scheme:

the porcine immature testis supporting cell line was cultured.

The invention firstly provides a recombinant plasmid for improving the lactic acid secretion level in a pig immature testis support cell line, which is obtained by inserting HSP90AA1 genes into pBI-CMV3 plasmid and sequencing the first generation of the full-length CDS sequence (2202 bp) of the pig HSP90AA1 is shown as SEQ ID NO. 1.

The preparation method of the specific recombinant plasmid comprises the following steps:

construction of an overexpression vector pBI-HSP90AA1 of porcine HSP90AA1. Molecular cloning and sequencing of porcine immature testis support cell HSP90AA1 gene, designing primers by referring to NM_213973.2 (https:// www.ncbi.nlm.nih.gov/nuccore/NM_ 213973.2) in NCBI, performing RT-PCR amplification on total RNA obtained from porcine immature testis support cell to obtain a full-length coding sequence of HSP90AA1, cloning into a T vector, and sequencing. The full length HSP90AA1 coding sequence was inserted into the multiple cloning site of the pBI-CMV3 empty vector (Clontech) via MluI and NotI endonucleases to construct the pBI-HSP90AA1 overexpression vector. The constructed pBI-HSP90AA1 over-expression vector is digested by MluI and NotI, and the size of the constructed vector is identified by agarose gel, as shown in figure 1, so that the recombinant plasmid can be obtained.

Amplification of the upstream primer sequences of HSP90AA1 cds: 5'-GAGAACGCGTATGCCCGAGGAA-3'

Amplification of the downstream primer sequences of HSP90AA1 cds: 5'-GAGAGCGGCCGCCTAATCGACTTC-3'.

The specific method for transfecting the overexpression HSP90AA1 plasmid is as follows:

by using

The HD (Promega) reagent transfects the over-expression vector into the pig immature testis support cells, the control group is empty vector pBI-CMV3, and the experimental group is pBI-HSP90AA1. Transfection was performed when the cell density reached around 80%.

The invention also provides a method for effectively detecting the lactic acid secretion level in the pig immature testis support cell line, wherein the HSP90AA1 is used as a marker gene for detecting the lactic acid secretion level in the pig immature testis support cell line, namely the expression level of the HSP90AA1 gene in the pig immature testis support cell line is detected. Specifically, the marker gene is applied by detecting HSP90AA1mRNA of the transfected pig immature testis support cells by RT-qPCR, detecting protein level of the transfected pig immature testis support cells by Western blot, detecting protein localization of the transfected pig immature testis support cells by cell immunofluorescence, or detecting lactic acid dehydrogenase activity and lactic acid level in cells after overexpression of HSP90AA1 by ELISA.

After 24h transfection, samples are collected, total RNA and protein are extracted, the levels of the over-expressed mRNA and protein are verified through RT-qPCR and western blot, the levels and the positions of the over-expressed protein are detected through immunofluorescence, and then the change of the over-expressed HSP90AA1 on the lactic acid level of the pig immature testis support cell is further detected through enzyme-linked immunosorbent assay.

Treatment of porcine immature testis-bearing cells with allolylamino-17-desmethoxygeldanamycin (17-AAG; abcam, cat# ab 141433) specifically inhibited HSP90AA1, control in DMSO solvent and experimental in 2. Mu.M 17-AAG. After 36h of treatment, samples are collected, total proteins are extracted, reduction of protein level of HSP90AA1 is verified through western blot, and then enzyme-linked immunosorbent assay is further used for inhibiting change of lactic acid level of HSP90AA1 on immature testis support cells of pigs.

Compared with the prior art, the invention has the beneficial effects that: the expression of HSP90AA1 can regulate the endogenous lactic acid production and metabolism level of the pig immature testis support cells, influence the functions of the cells, and provide a beneficial method for further researching the effect of the endogenous lactic acid production and metabolism of the cells on male reproduction in vitro.

The method is simple, and the secretion level of lactic acid in the immature testis support cells of the pigs is regulated by the expression of HSP90AA1 genes: after the HSP90AA1 gene of the pig immature testis support cell is over-expressed, the expression level of HSP90AA1mRNA and protein in the pig immature testis support cell is obviously increased, the activity of lactic acid dehydrogenase of the cell is obviously increased, and the level of lactic acid produced by the cell is obviously increased. After 17-AAG is treated on the pig immature testis support cells, the expression level of HSP90AA1 protein in the pig immature testis support cells is obviously reduced, the activity of lactic dehydrogenase of the cells is obviously reduced, and the level of lactic acid produced by the cells is obviously reduced.

Drawings

Fig. 1: constructing a swine HSP90AA1 overexpression vector, wherein FIG. 1A is an agarose gel electrophoresis diagram of a swine HSP90AA1 full-length coding sequence amplified by RT-PCR; FIG. 1B is a map of an empty expression vector of pBI-CMV 3; FIG. 1C is a map of a pBI-HSP90AA1 over-expression vector after cloning the full length coding sequence of porcine HSP90AA1 into a pBI-CMV3 expression vector; FIG. 1D shows agarose gel electrophoresis after cleavage to identify the size of the insert and empty vector;

fig. 2: detection of overexpression effect of HSP90AA1, FIG. 2A shows mRNA level of HSP90AA1 after transfection of pBI-HSP90AA1 for 24h by RT-qPCR, and FIG. 2B shows protein expression of HSP90AA1 after transfection of pBI-HSP90AA1 for 24h by Western Blot; FIG. 2C shows fluorescence intensity and localization of HSP90AA1 protein after 24h transfection of pBI-HSP90AA1 by cellular immunofluorescence assay; FIG. 2D is a graph showing the quantitative analysis of the cellular immunofluorescence intensity of HSP90AA1 protein after 24h transfection of pBI-HSP90AA 1: control, no transfection set; pBI-CMV3, transfected empty vector group; pBI-HSP90AA1, a group of transfected HSP90AA1 overexpression vectors; * P <0.05; * P <0.01; * P <0.001 (magnification: scale 100 μm);

fig. 3: enzyme-linked reaction detection of overexpression of HSP90AA1 to regulate lactate levels and lactate dehydrogenase activity, fig. 3A shows that overexpression of HSP90AA1 significantly increases the level of lactate production by porcine immature testis support cells; FIG. 3B is a graph showing that overexpression of HSP90AA1 significantly increases lactate dehydrogenase activity; * P <0.05;

fig. 4: 17-AAG-treated pig immature testis support cells, FIG. 4A is a photograph of a bright field (magnification: 500 μm scale) of 2. Mu.M 17-AAG-treated pig immature testis support cells for 36 hours; FIG. 4B is a graph showing that 2. Mu.M 17-AAG treated porcine immature testis support cells for 36h significantly reduced protein levels of HSP90AA 1; * P <0.05;

fig. 5: enzyme-linked reaction detection 17-AAG inhibits HSP90AA1 to regulate lactate level and lactate dehydrogenase activity, and FIG. 5A shows that after 2 mu M17-AAG treatment of immature testis support cells of pigs for 36 hours, the level of HSP90AA1 protein is inhibited, the level of cell lactic acid generation is obviously reduced; FIG. 5B is a graph showing that 2. Mu.M 17-AAG treated porcine immature testis support cells significantly reduced lactate dehydrogenase activity after 36h inhibition of HSP90AA1 protein levels; * P <0.01.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1: construction and transfection of HSP90AA1 overexpression vector

Referring to the HSP90AA1 sequence published on GenBank, 1 pair of primers was designed and synthesized to amplify the HSP90AA1 full length, upstream primer 5'-GAGAACGCGTATGCCCGAGGAA-3', downstream primer 5'-GAGAGCGGCCGCCTAATCGACTTC-3'. Using testis support cell cDNA as template, using 2X PCR Solution Premix Prime

HS enzyme amplifies HSP90AA1 gene fragment, and the size of the amplified fragment is expected to be 2202bp. PCR reaction conditions: denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 2min,30 cycles. And 1.0% agarose gel electrophoresis observation result, and judging whether the detection is accurate or not according to the fragment size. Cutting gel to recover target fragment, measuring concentration, double enzyme cutting, connecting T carrier at 16 deg.C overnight and converting competent large intestineAnd (3) bacillus, selecting positive clones for colony PCR identification, performing amplification culture on positive recombinants, and extracting plasmids for sequencing and comparison. The plasmid with correct sequence and empty plasmid pBI-CMV3 are subjected to double enzyme digestion, gel running, gel cutting recovery of target fragment, concentration measurement, overnight connection at 16 ℃, positive clone screening after conversion of the connection product, amplification culture, extraction of plasmid and double enzyme digestion verification of insertion of target gene. When the density of the porcine immature testis supporting cells reaches about 80%, transfection is started, and the whole transfection process is not added with double antibodies. By using

The HD reagent transfects plasmid, the specific reaction system is pBI-CMV3/pBI-HSP90AA1 plasmid 2 mug,

HD 7.5. Mu.L, serum free medium 100. Mu.L. The cells were transfected for 24h and were collected for use.

As shown in FIG. 1, the construction of the overexpression vector was successful, and the sequencing result was consistent with the cds sequence of porcine HSP90AA1 in the database.

Example 2: RT-PCR, western and cell immunofluorescence detection of over-expression effect

RT-qPCR: cell collection of untransfected control, transfected empty vector and transfected HSP90AA1 overexpression vector, total RNA extraction by Trizol, reverse transcription by ABI reverse transcription kit, preparation of qPCR by Roche FastStart Universal STBR Green Master (Rox) kit, and PCR reaction conditions are as follows: 50 ℃ 2min,95 ℃ 10min,95 ℃ 15s,60 s,40 cycles, upstream primer: 5'-ATCGCCCAGTTGATGTCGTT-3', downstream primer: 5'-GTGAGGGTCCGGTCTTGCT-3'. The expression level is calculated by using beta-actin as reference gene and using 2-delta Ct method, and SPSS software is used for statistical analysis.

Western: cells of the untransfected control group, the transfected empty vector group and the transfected HSP90AA1 overexpression vector group are collected, RIPA (containing PMSF, 1:100) lysate is lysed, supernatant is obtained by centrifugation, and the concentration is measured by a BCA method. Electrophoresis conditions 5% concentrated gel 80v,40 min; 12% separation gel 120V for 100 min. Transfer conditions were 200mA for 70 minutes. HSP90AA1 and beta-actin primary antibody dilution ratio is 1:1000, and secondary antibody dilution ratio is 1:10000.Image J analyzes the gray value.

Cellular immunofluorescence: cells from the untransfected control group, transfected empty vector group and transfected HSP90AA1 overexpressing vector group were fixed for 40min at room temperature, punched overnight in 1% Triton permeabilization solution, 1% BSA blocked for 1H at room temperature, 1% BSA diluted Rabbit Anti-HSP 90AA1 polyclonal antibody (1:50) incubated overnight at 4 ℃, washed 3 times in wash solution (PBS containing 0.01% Triton X-100,0.1% Tween-20), 1% BSA diluted secondary antibody (Goat Anti-Rabbit IgG (H & L)) at a ratio of 1:150 for 1H (light-shielding), hoechst33342 nuclei, anti-fluorescence quencher blocking, photographs observed under an inverted fluorescent microscope, scanning under a Lycra laser confocal microscope, and data analysis.

As shown in fig. 2, the mRNA and protein levels of cells transfected with HSP90AA1 overexpression vector were significantly increased in the experimental group compared to the non-transfected control group and the transfected empty vector group (< 0.05; P <0.01 and P < 0.001), indicating that the constructed over-expression vector can significantly increase the expression of HSP90AA1 in porcine immature testis supporting cell species.

Example 3: overexpression of HSP90AA1 can significantly improve activity of lactic dehydrogenase and lactic acid production of porcine immature testis support cells

Lactic acid measurement: is detected by a lactic acid detection kit. First, 20. Mu.L of cell supernatants of the empty vector group and the over-expression group were added to 5mL of EP tube, 1mL of working solution (prepared by an enzyme stock solution and an enzyme dilution solution at a ratio of 1:100) and 200. Mu.L of a color-developing agent were added to the EP tube, and after mixing, they were placed in a water bath at 37℃for reaction for 10min, 2mL of a stop solution was added to each tube, and the reacted sample was added to an ELISA plate at 200. Mu.L/well, and 3 replicates were performed for each group. And (3) detecting absorbance at the OD530nm of the enzyme-linked immunosorbent assay. All experimental conditions were kept consistent and three replicates were performed. The level of lactate secretion by the cells of the control and overexpressing groups was calculated from the standard.

Lactate dehydrogenase activity assay: is detected by a lactate dehydrogenase detection kit. Taking 20 mu L of cell supernatant of an empty vector group and an over-expression group, adding 25 mu L of matrix buffer solution and 5 mu L of coenzyme I, uniformly mixing, placing in a metal bath (37 ℃) for reaction for 15min, adding 25 mu L of 2, 4-dinitrophenylhydrazine, continuously placing in the metal bath (37 ℃) for reaction for 15min, adding 250 mu L of 0.4mol/L NaOH solution, and measuring absorbance value at an OD450nm position of an enzyme-linked immunosorbent assay. All experimental conditions were kept consistent and three replicates were performed. Control and overexpressed lactate dehydrogenase activities were calculated from the standards.

As shown in fig. 3: compared with an empty vector group, the lactic acid level and the lactic acid dehydrogenase activity of an experimental group transfected with the HSP90AA1 over-expression vector are obviously increased (P is less than 0.05), which indicates that the lactic acid secretion of the pig immature testis support cells can be obviously promoted after the expression of the HSP90AA1 is increased.

Example 4: 17-AAG-treated porcine immature testis supporting cells

After cells were cultured in a 5% CO2 cell incubator at 37℃until the density reached 30%, 17-AAG was added to the 17-AAG-treated group, 2. Mu.M 17-AAG was added to the DMSO-treated group, and DMSO was added in an equivalent volume to 17-AAG, and the incubator was incubated for 36 hours to collect cells, and HSP90AA1 protein levels were detected by referring to the western blot step described above.

As shown in FIG. 4, the protein level of HSP90AA1 in the experimental group of 2. Mu.M 17-AAG-treated 36h pig immature testis support cells was significantly less than that in the control group DMSO (< 0.05), indicating that 2. Mu.M 17-AAG treatment 36h significantly inhibited protein expression of HSP90AA1.

Example 5: inhibition of HSP90AA1 expression by 17-AAG can significantly reduce secretion of lactic acid by porcine immature testis cells

The culture supernatants of porcine immature testis support cells from the 2. Mu.M 17-AAG-treated group and DMSO control group were assayed for lactate levels and lactate dehydrogenase activity by reference to the experimental procedures above.

As shown in fig. 5, both lactate levels and lactate dehydrogenase activity were significantly lower in the experimental group 2 μm 17-AAG than in the control DMSO group (p < 0.01), indicating that inhibition of HSP90AA1 expression by 17-AAG down-regulates lactate production by porcine immature testis support cells.

The above-described embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.

SEQUENCE LISTING

<110> 1

<120> method for regulating lactic acid secretion level of pig immature testis support cell and application thereof

<130> university of Yangtze river

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 2202

<212> DNA

<213> porcine immature testis support cell HSP90AA1 full-length cds

<400> 1

atgcccgagg aaacccagac ccaagaccag ccgatggagg aggaggaggt ggagacgttc 60

gccttccagg cggaaatcgc ccagttgatg tcgttgatca tcaacacttt ctactcgaac 120

aaggagatct ttctgaggga gctcatttcc aactcgtccg atgctttgga caagatcaga 180

tacgaaagcc tgacggatcc cagtaaacta gactccggga aagagctgca cattaatctc 240

attccgaaca agcaagaccg gaccctcacg atagtggaca ccggcatcgg catgaccaag 300

gccgacttga tcaataacct tggtacgatc gccaagtctg ggaccaaggc gttcatggag 360

gctttgcagg ccggtgccga tatctcgatg attggccagt tcggtgtcgg cttctactct 420

gcgtacctgg tcgctgagaa agtgaccgtt atcaccaaac acaacgatga cgagcagtat 480

gcctgggagt cttctgcagg aggatctttc accgttagga cagacacagg agaacctatg 540

ggtcgtggaa caaaggttat tctacatctg aaagaagacc aaactgagta cttggaagaa 600

aggagaataa aggagattgt gaagaaacac tctcagttta ttggctaccc cattactctc 660

ttcgtggaga aggaacgtga taaagaagtc agtgacgacg aggcggaaga aaaggaagac 720

aaagaggaag aaaaggagaa agaagagaag gaatctgagg ataaaccgga gatagaagat 780

gttggttctg atgaagagga agaagaaaag aaggatggtg acaagaagaa gaagaagaag 840

atcaaggaga agtatattga tcaagaggaa ctcaacaaga caaagcctat ctggaccaga 900

aaccccgatg acatcactaa tgaagagtac ggggagttct ataagagctt gaccaatgac 960

tgggaggatc acttggctgt gaagcacttt tcagtcgaag ggcagttgga gttcagagcc 1020

cttcttttcg tcccaagacg cgctcctttc gacttatttg aaaacagaaa gaagaagaac 1080

aacatcaagc tgtatgttcg cagagtgttc atcatggaca actgcgagga gctcatccct 1140

gagtatctga atttcattag aggcgtggtg gactctgagg atcttcctct gaacatttct 1200

cgtgagatgt tgcaacaaag caaaattttg aaagtcatca ggaagaatct ggtcaagaaa 1260

tgcttggagc tctttactga attggccgaa gataaagaga attacaagaa gttctatgag 1320

cagttctcta aaaatattaa gcttggaata catgaagatt ctcaaaatcg gaagaagctt 1380

tccgagctgt tgaggtacta cacttctgct tctggcgacg agatggtttc cctcaaggac 1440

tattgcacca gaatgaagga gaaccagaaa cacatctatt acatcacagg tgagaccaag 1500

gaccaggtgg ccaactcggc cttcgtggaa cgtctgcgga agcacggcct ggaggtgatc 1560

tacatgatcg agcccatcga tgagtactgt gtgcagcagc tgaaggagtt tgaggggaag 1620

accttagtgt cagtcaccaa agagggcctg gagctcccgg aagatgaaga ggagaagaag 1680

aaacaggagg agaagaagac aaagtttgaa aacctctgca agatcatgaa ggacatcttg 1740

gagaagaaag tcgaaaaggt ggttgtgtcg aaccggttgg tgacctcacc gtgctgcatt 1800

gtcacaagca catacggctg gacagcaaac atggagagaa tcatgaaggc tcaagccctg 1860

agagacaact cgacaatggg ttacatggca gcaaagaagc acctggagat aaatccagac 1920

cattccatca tcgagaccct gaggcaaaag gcagaggcgg acaagaacga caagtccgtg 1980

aaggatctgg tcatcctgct gtacgaaacc gctctgctgt cttctggctt cagcctggaa 2040

gatccccaga cgcacgccaa caggatctac aggatgatca aacttggtct tggtattgat 2100

gaggacgacc ccaccgccga cgacagcagc gctgctgtga cggaggagat gccacccctg 2160

gaaggggacg acgacacgtc ccgcatggag gaagtcgatt ag 2202

Claims (4)

1. Use of a recombinant plasmid obtained by inserting an HSP90AA1 gene into a pBI-CMV3 plasmid, wherein the nucleotide sequence of the CDS of the HSP90AA1 gene is shown in SEQ ID No.1, for increasing the level of lactate secretion in a porcine immature testis support cell line.

2. Use of the recombinant plasmid according to claim 1 for increasing the level of lactic acid secretion in a porcine immature testis support cell line, characterized in that: the preparation of the recombinant plasmid comprises the following steps:

1) Designing a primer, carrying out RT-PCR amplification on total RNA obtained from a pig immature testis support cell to obtain a full-length coding sequence of HSP90AA1, cloning into a T vector, and sequencing;

2) The full-length coding sequence of HSP90AA1 is inserted into the multiple cloning site of pBI-CMV3 through MluI and NotI endonucleases to form a pBI-HSP90AA1 overexpression vector, namely a recombinant plasmid.

3. A method of increasing lactic acid secretion levels in a porcine immature testis support cell line, comprising: 1) Culturing a porcine immature testis support cell line; 2) Transfecting the recombinant plasmid of claim 1 into a pig immature testis support cell.

4. A method of modulating lactic acid secretion levels in a porcine immature testis support cell line, comprising: increasing the level of lactate secretion in a porcine immature testis support cell line by the method of claim 3, and decreasing the level of lactate secretion in a porcine immature testis support cell line by the HSP90 specific inhibitor 17-AAG.

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