CN115607689A

CN115607689A - Application of KLF7 gene in preparation of drug for reversing cell senescence

Info

Publication number: CN115607689A
Application number: CN202211252179.0A
Authority: CN
Inventors: 蔡晓琛; 顾雨春; 谭帅帅; 吴理达
Original assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Priority date: 2022-10-13
Filing date: 2022-10-13
Publication date: 2023-01-17

Abstract

The invention provides an application of KLF7 gene in preparing a medicine for reversing cell senescence. Compared with the prior art, the invention has the following advantages: the invention discovers that the KLF7 gene is related to cell senescence for the first time, and provides the application of the KLF7 gene in preparing a medicine for reversing cell senescence, an expression vector of the over-expressed KLF7 gene can effectively reverse senescence of key cells in an organism body, experiments prove that the over-expressed KLF7 gene can actually reverse cell senescence, further effective treatment of various diseases caused by organism senescence is realized, the effect is good, and cell experiments also prove that the proportion of non-senescent cells is effectively improved.

Description

Application of KLF7 gene in preparation of drug for reversing cell senescence

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a KLF7 gene in preparation of a medicine for reversing cell senescence.

Technical Field

With the increasing life span of people in modern society, the risk of people suffering from age-related diseases is also increasing. In fact, the data show that the greatest risk factor for heart disease, cancer and neurodegenerative disease is age, while the key to preventing or reversing aging is cell reprogramming. The Yamanaka afactors (Oct 4, sox2, klf4 and c-Myc transcription factors) can transform somatic cells into induced pluripotent stem cells after being introduced into mature somatic cells through virus vectors. Its discoverer was named "shanzhong zhimi", which was also awarded the nobel physiological or medical prize in 2012 for "finding four transcription factors with cellular reprogramming ability". In 2016, it was first reported that mountain factors could be used to reverse signs of aging in mice with premature aging disease and improve tissue function in the heart and brain. In addition, they found that the mountain midkine can accelerate muscle regeneration even in young mice. However, the effect of the mountain factor on physiologically senescent wild-type mice is not clear. Besides the mountain factors, the research on whether other factors or proteins can also produce similar or stronger effects on reversing aging can be realized, and the method has great social and economic effects.

Most cells have limited replication generations in vitro or in vivo, and can have cell aging phenomena under the action of replication of a certain generation or environmental oxidation factors, senescence promotion factors and the like, influence cell passage and metabolism in vitro, influence tissue repair and renewal in vivo, and show individual-level aging and a plurality of aging diseases along with age. Small molecule drugs for delaying aging include metformin, NMN, uridine, etc., but the action mechanism is unknown.

There are also a few reports in the literature of modifying the state of cells by driving epigenetic reprogramming against senescence-associated factors, for example, patent application CN112154210a discloses a transient cellular reprogramming method for reversing cellular aging, comprising transfecting the cells with one or more non-integrative messenger RNAs encoding one or more cellular reprogramming factors for no more than five consecutive days, thereby producing regenerated cells; the transcriptome profile of the regenerated cells is more similar to that of young cells, including increased expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, elf1, phf, pol2s2, taf and Sin3 a. However, the reprogramming method is complex in operation and poor in repeatability, and is not suitable for large-scale application.

Most of the existing researches focus on the mechanism/mechanism and the like of KLF7 in treating certain actual diseases, for example, the influence of KLF7 on apoptosis and proliferation of traumatic brain injury hippocampal neuron cell models reveals that KLF7 can inhibit apoptosis of TBI injury hippocampal neurons, and the mechanism of the mechanism is possibly related to the regulation of a JAK2/STAT3 signal pathway; the research on the function and molecular mechanism of KLF7 in the process of promoting the generation and development of prostate cancer by FFAs reveals that the high expression of KLF7 is closely related to the generation and development of PCa related to obesity, IL-6 and p21 can be downstream target genes of KLF7, and the KLF7 can promote the biological behavior of PCa cells by up-regulating the expression of IL-6 and down-regulating the expression of p 21; the research on the function and the primary mechanism of KLF7 in macrophage inflammatory reaction of epicardial adipose tissue of coronary heart disease reveals that the expression of KLF7 is increased in EAT of CAD patients, and the KLF7 mediates the release of inflammatory factors in EAT by regulating and controlling NF-kB inflammatory signal pathways.

At present, no literature reports of the use of KLF7 in association with and reversal of cellular senescence have been found.

Disclosure of Invention

Aiming at the technical limitations, the invention discovers that the KLF7 gene is related to cell senescence for the first time, and the overexpression of the gene can reverse the cell senescence so as to treat senescence-related diseases, which has never been reported in the prior art; based on the discovery, the invention provides the application of the KLF7 gene in preparing a medicament for reversing cell aging, and overcomes the defects in the background art.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention provides application of a KLF7 gene in preparing a medicament for reversing cell senescence.

Optionally, in the above application, the nucleotide sequence of the KLF7 gene is shown as SEQ ID No.1, and the amino acid sequence of the KLF7 gene encoded protein is shown as SEQ ID No. 2.

The nucleotide sequence SEQ ID No.1 of the KLF7 gene is:

ATGGACGTGTTGGCTAGTTATAGTATATTCCAGGAGCTACAACTTGTCCACGACACCGGCTACTTCTCAGCTTTACCATCCCTGGAGGAGACCTGGCAGCAGACATGCCTTGAATTGGAACGCTACCTACAGACGGAGCCCCGGAGGATCTCAGAGACCTTTGGTGAGGACTTGGACTGTTTCCTCCACGCTTCCCCTCCCCCGTGCATTGAGGAAAGCTTCCGTCGCTTAGACCCCCTGCTGCTCCCCGTGGAAGCGGCCATCTGTGAGAAGAGCTCGGCAGTGGACATCTTGCTCTCTCGGGACAAGTTGCTATCTGAGACCTGCCTCAGCCTCCAGCCGGCCAGCTCTTCTCTAGACAGCTACACAGCCGTCAACCAGGCCCAGCTCAACGCAGTGACCTCATTAACGCCCCCATCGTCCCCTGAGCTCAGCCGCCATCTGGTCAAAACCTCACAAACTCTCTCTGCCGTGGATGGCACGGTGACGTTGAAACTGGTGGCCAAGAAGGCTGCTCTCAGCTCCGTAAAGGTGGGAGGGGTCGCAACAGCTGCAGCAGCCGTGACGGCTGCGGGGGCCGTTAAGAGTGGACAGAGCGACAGTGACCAAGGAGGGCTAGGGGCTGAAGCATGTCCCGAAAACAAGAAGAGGGTTCACCGCTGTCAGTTTAACGGGTGCCGGAAAGTTTATACAAAAAGCTCCCACTTAAAGGCCCACCAGAGGACTCACACAGGTGAGAAGCCTTATAAGTGCTCATGGGAGGGATGTGAGTGGCGTTTTGCACGAAGCGATGAGCTCACGAGGCACTACAGGAAACACACAGGTGCAAAGCCCTTCAAATGCAACCACTGCGACAGGTGTTTTTCCAGGTCTGACCATCTTGCCCTCCACATGAAGAGACATATCGGATCCGGC。

the amino acid sequence SEQ ID No.2 of the protein coded by the KLF7 gene is:

MDVLASYSIFQELQLVHDTGYFSALPSLEETWQQTCLELERYLQTEPRRISETFGEDLDCFLHASPPPCIEESFRRLDPLLLPVEAAICEKSSAVDILLSRDKLLSETCLSLQPASSSLDSYTAVNQAQLNAVTSLTPPSSPELSRHLVKTSQTLSAVDGTVTLKLVAKKAALSSVKVGGVATAAAAVTAAGAVKSGQSDSDQGGLGAEACPENKKRVHRCQFNGCRKVYTKSSHLKAHQRTHTGEKPYKCSWEGCEWRFARSDELTRHYRKHTGAKPFKCNHCDRCFSRSDHLALHMKRHI。

optionally, for the above applications, the cell comprises human umbilical vein endothelial cell, human neuroblastoma cell, fibroblast cell, and mesenchymal stem cell.

The expression vector of over-expressing KLF7 gene prepared by the invention can be expected to be used for preparing a medicine for treating related diseases caused by cell aging.

The diseases caused by cell aging include atherosclerosis, cardiovascular diseases, cachexia, arthritis, cataract, osteoporosis, diabetic retinopathy, hypertension, neurodegenerative diseases, apoplexy/cerebral apoplexy, atrophic gastritis, trunk precursor disease, chronic obstructive pulmonary disease, coronary artery disease, dopamine dysregulation syndrome, metabolic syndrome, hashimoto thyroiditis, heart failure, senile depression, myocardial infarction, acute coronary syndrome, sarcopenia, senile obesity, senile osteoporosis, urinary incontinence and natural aging.

The related diseases caused by cell aging are preferably cataract, diabetic retinopathy, and natural aging.

Optionally, the application is that the KLF7 gene is inserted into a skeleton vector to construct an expression vector over-expressing the KLF7 gene; then packaging the expression vector of the over-expression KLF7 gene by using adeno-associated virus to obtain the adeno-associated virus of the over-expression KLF7 gene; finally, the adeno-associated virus over-expressing KLF7 gene is transfected into the cells or directly injected into human or animal bodies.

Optionally, for the above application, the backbone vector is pSFFV-T2A-mCherry plasmid vector.

Optionally, in the above application, the KLF7 gene overexpression expression vector is constructed by inserting the KLF7 gene sequence between pSFFV and T2A of the backbone vector pSFFV-T2A-mCherry plasmid by a homologous recombination method, and constructing an overexpression vector pSFFV-KLF7-T2A-mCherry plasmid.

Optionally, for the above-described use, the adeno-associated virus is an AAV vector of AAV2/2 serotype or an AAV vector of AAV2/9 serotype.

Alternatively, for the above-described use, the reversal of cellular senescence is manifested in a reduction in the proportion of cells that stain positively for β -galactosidase.

Compared with the prior art, the invention has the following advantages:

the invention discovers that the KLF7 gene is related to cell senescence for the first time, and provides the application of the KLF7 gene in preparing a medicine for reversing cell senescence, an expression vector of the over-expressed KLF7 gene can effectively reverse senescence of key cells in an organism body, experiments prove that the over-expressed KLF7 gene can actually reverse cell senescence, further effective treatment of various diseases caused by organism senescence is realized, the effect is good, and cell experiments also prove that the proportion of non-senescent cells is effectively improved.

Drawings

FIG. 1 shows the plasmid vector structure of pSFFV-KLF7-T2A-mCherry.

FIG. 2 shows the morphology under a light microscope after staining HUVEC cells transfected with KLF7 gene and control cells, showing that transfection of KLF7 is effective in reversing HUVEC cell senescence.

FIG. 3 shows the ratio of non-senescent cells detected after KLF7 was transferred into SH-SY5Y human neuroblastoma cells and a control.

FIG. 4 is a diagram showing the results of fibroblast senescence caused by the inversion of the KLF7 gene.

FIG. 5 is a graph showing the results of reversing mesenchymal stem cell senescence by electrotransfering the KLF7 gene.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It is to be understood that the description herein is only illustrative of the present invention and is not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention. The reagents and instruments used in the present invention are commercially available, and the characterization means involved can be referred to the description in the prior art, which is not repeated herein.

For a further understanding of the present invention, reference will now be made in detail to the preferred embodiments of the present invention.

Example 1

Application of KLF7 gene in preparing medicine for reversing cell senility.

The nucleotide sequence of the KLF7 gene is shown as SEQ ID No.1, and the amino acid sequence of the KLF7 gene coding protein is shown as SEQ ID No. 2.

The nucleotide sequence SEQ ID No.1 of the KLF7 gene is:

the amino acid sequence SEQ ID No.2 of the protein coded by the KLF7 gene is:

the cells comprise human umbilical vein endothelial cells, human neuroblastoma cells, fibroblasts and mesenchymal stem cells.

The application is that KLF7 gene is inserted into a skeleton vector to construct an expression vector over-expressing the KLF7 gene; then packaging an expression vector for over-expressing the KLF7 gene by using the adeno-associated virus to obtain the adeno-associated virus for over-expressing the KLF7 gene; finally, the adeno-associated virus which over-expresses KLF7 gene is transfected into the cells or directly injected into human or animal bodies.

The skeleton vector is pSFFV-T2A-mCherry plasmid vector.

The construction method of the expression vector for over-expressing the KLF7 gene comprises the steps of inserting the KLF7 gene sequence between pSFFV and T2A of a skeleton vector pSFFV-T2A-mCherry plasmid by adopting a homologous recombination method, and constructing the over-expression vector pSFFV-KLF7-T2A-mCherry plasmid.

The adeno-associated virus is AAV2/2 serotype AAV vector or AAV2/9 serotype AAV vector.

Reversing cellular senescence is manifested in a reduction in the proportion of cells that stain positive for β -galactosidase.

Example 2

Vector construction:

1. the pSFFV-OSK-T2A-mCherry plasmid is taken as a skeleton vector for gene expression;

2. the gene sequence is synthesized, the gene sequence of KLF7 and the like is inserted between pSFFV and T2A of pSFFV-T2A-mCherry plasmid by a homologous recombination method, and the vector is named as pSFFV-KLF7-T2A-mCherry.

The plasmid vector structure of pSFFV-KLF7-T2A-mCherry is shown in FIG. 1.

Example 3

1. Packaging of adeno-associated virus:

1. cell inoculation:

t175 Flasks inoculation 2X 10 ⁷ And 293FT cells. Adding 30ml of 10-percent FBS-containing DMEM medium, 37 ℃,5% ₂ The incubator is used for overnight culture, and transfection is carried out after 16-24 h.

2. Cell transfection:

the confluence of cell growth reaches 80-90%, and transfection is prepared. The transfection system is shown in Table 1 below.

TABLE 1

Mixing solution A and solution B, and standing at room temperature for 5min. And then dropwise adding the solution B into the solution A while shaking uniformly, and standing at room temperature of 22-26 ℃ for 20min. Adding into culture dish drop by drop, shaking gently, and adding 5% CO ₂ The cells were cultured overnight at 37 ℃.

3. And (3) transfection and liquid change:

16-18h later, the medium containing the transfection reagent was removed, 30ml of DMEM containing 10% FBS was added, 5% CO ₂ The culture was continued at 37 ℃.

4. Harvesting the virus for the first time:

48h after the start of transfection, the cell supernatants were harvested, transferred to 50ml centrifuge tubes, filtered through 0.45 μm filters and stored at 4 ℃. The cells were added to 30ml of DMEM containing 10% FBS, 5% CO ₂ The culture was continued at 37 ℃.

5. And (3) harvesting the virus for the second time:

the cell supernatant was harvested, transferred to a 50ml centrifuge tube, filtered through a 0.45 μm filter and stored at 4 ℃. The cells were treated with 10% disinfectant (84 disinfectant) and discarded.

6. And (3) virus concentration:

filtering the collected adeno-associated virus component by using a 0.45 mu m filter to remove bacterial contamination, and mixing the filtered component with PEG8000 according to a volume ratio of 3:1, mixing and mixing by gently inverting.

7. Incubate at 4 ℃ for 30min or overnight.

8. Centrifugation at 1500g for 45min at 4 ℃ resulted in a white precipitate at the bottom of the tube.

9. The supernatant was carefully aspirated without destroying the white precipitate.

10. Resuspending the precipitate with an appropriate volume of adeno-associated virus preservation solution, performing qPCR detection on the virus to determine the virus titer, and separately packaging and preserving the obtained adeno-associated virus at-80 ℃.

2. And (3) virus titer determination:

qPCR assay for adeno-associated virus to determine viral titer:

1. diluting the standard plasmid of virus to be detected by 10-fold gradient, and selecting 10 ¹¹ ～10 ⁷ copies/. Mu.L was used as a standard for the experimental standard curve.

2. According to the following

The Top Green qPCR Supermix instruction and the fluorescent quantitative PCR instrument reaction system require the preparation of qPCR reaction reagents, and the reaction system is shown in the following table 2.

TABLE 2

Composition (I)	Volume of
		Forward primer (10 μm)	1μL
Reverse primer (10 μm)	1μL
		2×TransStar Top/Tip Green qPCR SuperMix	10μL
Nuclease-free water	7μL
		DNA	1μL
Total volume	20μL

The sequence of the forward primer is GGAGTTGTGGCCCGTTGT;

the reverse primer sequence is GAGCCCCTGTCCAGCAGC.

3. And (3) subpackaging the reaction mixed solution into 8-connection tubes by taking 19 mu L/hole (20 mu L system), and sequentially adding 1 mu L/hole reaction standard substance and a sample to be detected into the holes.

4. Instantly separating the 8-connection tube added with the reaction solution, slightly shaking and uniformly mixing, and placing the mixture after the instantaneous separation

In the 96System Instrument, a reaction program was set and run. The reaction sequence is shown in table 3.

TABLE 3

5. And after the reaction program is finished, taking out the detection 8 connected tubes for discarding, copying data, and storing and analyzing the data.

3. The experimental results are as follows:

after the KLF7 is transferred into the adeno-associated virus, the AAV2/2-KLF7 virus titer is 1.3 multiplied by 10 ¹² 。

Example 4

HUVEC cell culture and electrotransformation:

1. experimental materials:

the details are shown in Table 4.

TABLE 4

Name (R)	Company(s)	Goods number
			Endothelial cell culture medium	ScienCell	BNCC342473
Human umbilical vein endothelial cell HUVEC	ScienCell	#8000
			Neon ^TM Initial set of transfection system	Thermo Fisher	MPK5000S
DPBS (calcium and magnesium free)	Gibco	2380005
			0.25% pancreatin	Cytiva	J210027
Cell aging beta-galactosidase staining kit	Biyuntian (a Chinese character)	C0602

2. The experimental method comprises the following steps:

(1) HUVEC cell recovery:

1) The 1 vial was removed from the liquid nitrogen tank and immediately shaken continuously in water at 37 ℃ to completely thaw the frozen cell suspension.

2) Once the cells in the cryopreservation tube are completely thawed (liquid), the cells are immediately taken out of the hot water bucket, 75% of alcohol is used for completely disinfecting the surface of the cryopreservation tube, and the cryopreservation tube is placed in an ultra-clean workbench. When the frozen tube is about to melt, the centrifuge tube containing the endothelial culture medium preheated to 37 ℃ is sterilized by alcohol and then is placed in an ultra-clean workbench.

3) Taking out the cell suspension in the frozen tube under strict aseptic operation conditions, adding the cell suspension into a preheated E8 culture medium 15ml centrifugal tube, gently blowing and beating for 2-3 times, centrifuging at 1300rpm for 5min, and discarding the supernatant after the centrifugation is finished.

4) Adding endothelial culture medium, gently blowing and beating for 2-3 times, transferring cells to cell culture flask, and adding culture solution. Placing the culture flask in CO ₂ Culturing in an incubator.

(2) HUVEC digestion passage:

1) The well plate/flask to be passaged was removed from the incubator, the supernatant was aspirated and washed once with DPBS.

2) Adding trypsin, spreading the solution at the bottom of the bottle, removing trypsin, incubating in an incubator for 4-5min, and observing under a microscope to make the cells contract and become round and disperse.

3) Gently tapping the culture flask/plate to make the cells come off the wall, gently blowing and beating for several times with a gun head, adding endothelial culture medium to stop digestion, transferring the cells after being blown and beaten to a new culture flask, adding endothelial culture medium, standing at 37 deg.C and 5% CO ₂ Culturing in an incubator.

4) And (4) performing liquid change operation every three days, and performing passage when the cell confluence is about 70-80%.

(3) HUVEC cell electrotransformation:

HUVEC cells of passage 6, 20 ten thousand cells 1. Mu.g of plasmid were used.

Electric transfer conditions: voltage: 1350v, pulse time: 30ms, pulse number: 1;

after 3 days of electrotransfer, the liquid was changed, and after 9 days, the aged cells were counted.

(4) And (3) staining the aged cells:

HUVEC cells were stained using a beta-galactosidase staining kit.

a. For cells cultured in 24-well plates, the cell culture medium was aspirated, washed 1 time with DPBS, and fixed for 15 minutes at room temperature by adding 0.5 ml of β -galactosidase staining fixative. For other types of plates, the amounts of fixative and subsequent solutions are manipulated with reference to this ratio.

b. The cell fixative was aspirated and the cells were washed 3 times with DPBS for 3 minutes each.

c. PBS or HBSS was aspirated and 1ml of staining solution was added to each well. The preparation method of the dyeing working solution is shown in table 5.

TABLE 5

Dyeing working solution component	Volume of dose
		Beta-galactosidase staining solution A	10μl
Beta-galactosidase staining solution B	10μl
		Beta-galactosidase staining solution C	930μl
Beta-galactosidase chromogenic substrate X-Gal solution	50μl
		Total of	1000μl(1ml)

d. Incubation was overnight at 37 ℃ and 6-well plates were sealed with parafilm or cling film to prevent evaporation. Incubation at 37 ℃ cannot be performed in a carbon dioxide incubator.

e. And (4) observing under a common optical microscope.

3. The experimental results are as follows:

as shown in FIG. 2, the senescent cells appeared blue, and the proportion of senescent cells in HUVEC cells into which KLF7 gene was transferred was significantly less than that in HUVEC cells of the control group. Indicating that the transferred KLF7 gene can delay the senescence of HUVEC cells.

Example 5

SH-SY5Y (human neuroblastoma cell) senescent cell in vivo labeling and flow analysis:

1. experimental materials:

the details are shown in Table 6.

TABLE 6

Name (R)	Company(s)	Goods number
			MEM medium	Invitrogen	11090081
F12 Medium	Invitrogen	11765054
			Fetal bovine serum FBS	Gibco	—
Gluta-max additive	Invitrogen	35050061
			Pyruvic acid sodium salt	Invitrogen	11360070
Non-essential amino acid NEAA	Invitrogen	11140050
			SH-SY5Y	China academy of sciences (academy of sciences) type culture collection committee cell bank/stem cell bank	SCSP-5014
C12FDG reactive dyes	Abcam	138777-25-0
			Dimethyl sulfoxide DMSO	Sigma	D2650
Barveromycetin A1	Abcam	88899-55-2
			DPBS buffer (calcium and magnesium free)	Gibco	2380005

2. The experimental method comprises the following steps:

SH-SY5Y medium formula (100 ml): 43.5ml of MEM medium, 43.5ml of F12 medium, 10ml of fetal bovine serum FBS, 1ml of Gluta-max additive, 1ml of sodium pyruvate and 1ml of non-essential amino acid.

(1) Induction of SH-SY5Y senescence: using 150 μ M H ₂ O ₂ Replacing a normal culture medium for culturing for 24 hours after SH-SY5Y 2 hours;

(2) Treating the cells with 100 mu M Bafilomycin (Bafilomycin) for 1h, inhibiting the activity of beta-galactosidase in lysosomes, adding an active fluorescent substrate dye of beta-galactosidase into SH-SY5Y culture medium, culturing for 2h at 37 ℃, washing twice with DPBS, digesting with pancreatin, collecting the cells, and performing flow cytometry analysis. Comparison of the effects of over-expressed genes on reversal of senescence was made from mean fluorescence intensity and the proportion of cells stained less.

3. The experimental results are as follows:

as shown in FIG. 3, the non-senescent cell ratio after transferring KLF7 into SH-SY5Y was 41.7%, which was significantly higher than 18.6% of the control group and 17.5% of SH-SY5Y transferred into TP53 gene, and also higher than 31.3% of SH-SY5Y transferred into TP53 and KLF7 gene simultaneously. It is shown that at least on SH-SY5Y, the over-expression of KLF7 gene can delay cell aging and maintain the young state.

Example 6

Fibroblast culture and electrotransformation:

1. experimental materials:

specifically, the results are shown in Table 7.

TABLE 7

Name(s)	Company(s)	Goods number
			DMEM high-sugar medium	Gibco	12100-046
fetal bovine serum	Cellma	SA211.02
			Neon ^TM Initial set of transfection system	Thermo Fisher	MPK5000S
DPBS (calcium and magnesium free)	Gibco	2380005
			0.25% pancreatin	Cytiva	J210027
Cell aging beta-galactosidase staining kit	Biyuntian (a Chinese character)	C0602

2. The experimental method comprises the following steps:

(1) And (3) recovering the fibroblasts:

4) Adding endothelial culture medium, and gently blowingBeating for 2-3 times, transferring the cells into a cell culture bottle, and adding culture solution. Placing the culture flask in CO ₂ Culturing in an incubator.

(2) And (3) digesting and passaging fibroblasts:

4) And (4) performing liquid changing operation every three days, and performing passage when the cell confluence is about 70-80%.

(3) And (3) electrically transforming fibroblasts:

the 24 th generation cells, 20 ten thousand cells, 1. Mu.g of plasmid were used.

And (3) electrotransfer conditions: voltage: 1350v, pulse time: 30ms, pulse number: 1, changing the liquid after 3 days after the electrotransfer, and carrying out aging cell statistics after 9 days.

(4) And (3) staining the aged cells:

fibroblasts were stained using a beta-galactosidase staining kit.

c. PBS or HBSS was aspirated and 1ml of staining solution was added to each well. The dyeing working solution was prepared as shown in Table 5 of example 4.

d. Incubation was overnight at 37 ℃ and 6-well plates were sealed with parafilm or cling film to prevent evaporation. Note that: incubation at 37 ℃ cannot be performed in a carbon dioxide incubator.

e. And (4) observing under a common optical microscope.

3. The experimental results are as follows:

the staining results of beta-galactosidase are shown in FIG. 4, the senescent cells appeared blue, and the proportion of non-senescent cells in the fibroblasts transfected with the KLF7 gene was significantly higher than that of the control fibroblasts. Thus indicating that the transferred KLF7 gene can delay the aging of fibroblasts.

Example 7

Culturing mesenchymal stem cells and performing electrotransformation:

1. experimental materials:

specifically, the results are shown in Table 8.

TABLE 8

2. The experimental method comprises the following steps:

(1) Recovering the mesenchymal stem cells:

2) Once the cells in the cryopreservation tube are completely thawed (liquid), the cells are immediately taken out of the hot water bucket, 75% of alcohol is used for completely disinfecting the surface of the cryopreservation tube, and the cryopreservation tube is placed in an ultra-clean workbench. When the frozen tube is about to melt, the centrifuge tube containing the endothelial culture medium preheated to 37 ℃ is sterilized by alcohol and then placed in an ultra-clean workbench.

4) Adding endothelial culture medium, lightly blowing and beating for 2-3 times, transferring the cells to a cell culture bottle, and adding culture solution. Placing the culture flask in CO ₂ Culturing in an incubator.

(2) Digesting and passaging the mesenchymal stem cells:

2) Adding trypsin, spreading to the bottom of the bottle, sucking away the trypsin, incubating in an incubator for 4-5min, and observing under a mirror during the incubation period until the cells shrink and become round and disperse.

(3) Electrically transforming the mesenchymal stem cells:

the 3 rd generation cells, 20 ten thousand cells, 1. Mu.g of plasmid were used.

(4) Inducing MSC senescence:

MSCs were treated with 600nM doxorubicin for 2 days.

(5) And (3) staining the aged cells:

the mesenchymal stem cells were stained using a beta-galactosidase staining kit.

d. Incubation at 37 ℃ overnight, 6-well plates can be sealed with parafilm or plastic wrap to prevent evaporation, where incubation at 37 ℃ cannot be performed in a carbon dioxide incubator.

e. And observing under a common optical microscope.

3. The experimental results are as follows:

the staining result of beta-galactosidase is shown in figure 5, the senescent cells are blue, and the proportion of non-senescent cells in the mesenchymal stem cells with the KLF7 gene transferred into the mesenchymal stem cells is obviously higher than that of the mesenchymal stem cells in the control group. The fact that the KLF7 gene is transferred into the mesenchymal stem cells can delay the senescence of the mesenchymal stem cells is shown.

Example 8

Marking the aging cells of the mesenchymal stem cells in vivo and analyzing by flow:

1. experimental materials

Specifically, the results are shown in Table 9.

TABLE 9

Name (R)	Company (SA)	Goods number
			alpha-MEM Medium	Hyclone	SH30265.01
Z41	HPCPLCRL05	F5427
			C12FDG reactive dyes	Abcam	138777-25-0
DMSO	Sigma	D2650
			Bafilomycin A1	Abcam	88899-55-2
DPBS (calcium magnesium free)	Gibco	2380005

2. The experimental method comprises the following steps:

treating cells with 100 μ M Bafilomycin for 1h, inhibiting beta-galactosidase activity in lysosomes, adding an active fluorescent substrate dye of beta-galactosidase into an alpha-MEM medium, culturing at 37 ℃ for 2h, washing twice with DPBS, digesting with pancreatin, collecting cells, and performing flow cytometry analysis. Comparison of the effects of overexpression of the gene on reversal of senescence was made from the mean fluorescence intensity and the reduction of the cell proportion by staining.

3. The experimental results are as follows:

after KLF7 is transferred into mesenchymal stem cells, the proportion of non-aged cells is 19.3 percent and is obviously higher than 12.4 percent of that of a control group. The fact that the KLF7 gene is transferred into the mesenchymal stem cells can delay the senescence of the mesenchymal stem cells is shown.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

Application of KLF7 gene in preparing medicine for reversing cell senility.
2. The use according to claim 1, characterized in that the nucleotide sequence of the KLF7 gene is shown as SEQ ID No.1 and the amino acid sequence of the protein encoded by the KLF7 gene is shown as SEQ ID No. 2.
3. The use of claim 1, wherein the cells comprise human umbilical vein endothelial cells, human neuroblastoma cells, fibroblasts, mesenchymal stem cells.
4. The use of claim 1, wherein the use is the insertion of the KLF7 gene into a backbone vector to construct an expression vector overexpressing the KLF7 gene; then packaging the expression vector of the over-expression KLF7 gene by using adeno-associated virus to obtain the adeno-associated virus of the over-expression KLF7 gene; finally, transfecting the adeno-associated virus with over-expression KLF7 gene into the cell of claim 3 or directly injecting the cell into a human or animal body.
5. The use of claim 4, wherein the backbone vector is a pSFFV-T2A-mCherry plasmid vector.
6. The use of claim 4 or 5, wherein the expression vector for over-expressing KLF7 gene is constructed by inserting the KLF7 gene sequence between pSFFV and T2A of the backbone vector pSFFV-T2A-mCherry plasmid by homologous recombination to construct the over-expression vector pSFFV-KLF7-T2A-mCherry plasmid.
7. The use according to claim 4, wherein the adeno-associated virus is an AAV vector of serotype 2/2 AAV or an AAV vector of serotype 2/9 AAV.
8. The use according to claim 1, wherein said reversal of cellular senescence is manifested in a reduction of the proportion of cells staining positive for β -galactosidase.