CN106957820B - Culture medium for in vitro chondrocyte telomere prolonged proliferation culture, kit and application - Google Patents

Abstract

The invention discloses a culture medium for in vitro chondrocyte telomere prolonged proliferation culture, a kit and application. The culture medium for the in vitro chondrocyte telomere prolonged proliferation culture is prepared by adding serum, vitamin C with the final concentration of 5-50 mu g/ml, B18R or p65i with the final concentration of 0.01-2 mu g/ml, rapamycin with the final concentration of 0.1-50ng/ml and resveratrol with the final concentration of 0.1-50 mu M into a DMEM/F12 or RPMI1640 basic culture medium, wherein the serum is human AB serum with the volume final concentration of 1-20% or fetal bovine serum with the volume final concentration of 1-20%. By using the culture medium or the kit for in vitro chondrocyte telomere extension proliferation culture, telomerase mRNA transfection can be carried out on chondrocytes in vitro, so that telomeres in the chondrocytes obtained by subsequent culture are extended, the proliferation and the activity of the chondrocytes, particularly chondrocytes of old patients, can be obviously improved, and the culture medium or the kit for in vitro chondrocyte telomere extension proliferation culture can be practically applied to regenerative medical treatment for improving or treating cell function decline caused by aging.

Description

Culture medium for in vitro chondrocyte telomere prolonged proliferation culture, kit and application

Technical Field

The invention relates to the field of cell biology, in particular to a culture medium for in-vitro chondrocyte telomere prolonged proliferation culture, a kit and application.

Background

Telomeres are protective caps of chromosome ends, and are special structures composed of short multiple repeats of non-transcribed sequences (TTAGGG) and some binding proteins, so that the stability of a genome can be protected, and genetic materials at the chromosome ends can be prevented from being lost. The progressive expansion of cells progressively affects telomere length. When telomeres are shortened to a certain extent, their protection will disappear, which in turn causes aging and apoptosis of the cells.

Telomerase is an enzyme that synthesizes telomeres and maintains their length. Telomerase consists of two parts, an RNA template subunit, telomerase mRNA component, and a protein catalytic unit, telomerase reverse transcriptase. Not all somatic cells have telomerase. Embryonic cells actively express telomerase, but when the embryonic cells differentiate to some extent, the expression of telomerase is stopped. Only a few cells in adults have been reported to express telomerase, such as spermatogonia, activated T cells and some stem cells. The gene defect of telomerase can cause diseases such as presenile diseases, and patients have symptoms such as bone marrow failure and mucosa diseases, including abnormal pigmentation, nail malnutrition, oral leukoplakia and the like. Research has proved that after the telomerase is reintroduced into the model mouse with the telomerase deficiency, the aging symptoms tend to be corrected, including brain diseases, infertility and the like. Because of the relationship between telomeres and cell senescence, the telomere theory also becomes one of many senescence theories such as oxidative stress, saccharification, genes and the like, and becomes a hot topic in anti-senescence research.

The traditional DNA recombination technology for introducing telomerase gene into cell often involves plasmid and virus. Bodar et al transfected human cells that were almost devoid of telomerase activity with vectors containing the telomerase gene and found significant lengthening of telomeres, faster cell separation, and slower cell senescence (Bodar (1998) science.279: 349-352). The gene engineering product has high requirements on safety, including removal of contaminants such as infectious microbes, endotoxin, plasmids and the like, determination of virus self-replication capacity and the like, so that clinical use of the gene engineering product is limited; secondly, random insertion of genes can be caused, and instability of the genome can be caused; finally, stable expression of telomerase carries the risk of immortalizing the cells. These adverse factors affect the clinical use and marketing of telomerase gene therapy.

In recent years, introduction of messenger ribonucleic acid (mRNA) molecules to allow cells to express genes has been studied as a new means of gene therapy. Since mRNA is used directly in the cytoplasm as a template for protein synthesis, mRNA is not integrated into the genome after introduction into the cell and thus affects the stability of the genetic material. mRNA is a genetic material owned by human being itself and is safer than viruses. mRNA therapy has become a popular study, especially as influenza and tumor vaccines. The mRNA is capable of eliciting strong protective immunity against influenza virus for a long period of time. mRNA has also been tested in phase I/II clinical trials as a tumor vaccine, and most patients develop an immune response against tumor antigens.

With the improvement of the quality of life of people, more and more people join the exercise and fitness line, and the problem is that more and more people suffer from sports injuries, wherein the cartilage injuries, particularly the cartilage injuries of the knee joint and the ankle joint, account for a large proportion. According to the domestic and foreign literature reports, the incidence rate of cartilage damage in the knee arthroscopic population is between 61% and 68%. Patients often feel pain and stiffness, and finally become severe arthritis, even can not walk, and the quality of life is seriously affected.

The mature cartilage tissue is a nerve-free, blood vessel-free and lymphatic vessel-free tissue, and the chondrocytes are sparsely distributed in the cartilage tissue, so that the self-renewal speed of the cartilage tissue is slow, and the self-repair capacity is limited. Especially, the proliferation speed and the self-repairing capability of the chondrocytes of the elderly patients are very low, so that the application of the treatment of the aged cartilage injury is greatly limited. Therefore, the finding of a suitable method for increasing the number of times of chondrocyte population multiplication in vitro, especially in elderly patients, delaying cell aging, increasing cell activity, and avoiding the possibility of cell immortalization is a difficult point and key point for cartilage repair treatment.

Disclosure of Invention

Based on the above, there is a need for a culture medium, a kit and an application for in vitro chondrocyte telomere elongation proliferation culture, so as to safely and effectively prevent, improve or treat chondrocyte function deterioration caused by aging or injury, and can be applied to regenerative medicine treatment.

The technical scheme of the invention for solving the technical problems is as follows.

A culture medium for in vitro chondrocyte telomere prolonged proliferation culture is prepared by adding serum, vitamin C with a final concentration of 5-50 mu g/ml, B18R or p65i with a final concentration of 0.01-2 mu g/ml, rapamycin with a final concentration of 0.1-50ng/ml and resveratrol with a final concentration of 0.1-50 mu M into a DMEM/F12 or RPMI1640 basic culture medium, wherein the serum is human AB serum with a volume final concentration of 1-20% or fetal bovine serum with a volume final concentration of 1-20%.

In one embodiment, the serum in the culture medium is human AB serum at a final concentration of 5% -10% by volume or fetal bovine serum at a final concentration of 10% -20% by volume;

the final concentration of the vitamin C is 20-40 mug/ml;

the final concentration of the B18R or p65i is 0.1-0.5 μ g/ml;

the final concentration of the rapamycin is 1-10 ng/ml;

the final concentration of resveratrol is 1-10 μ M.

Further, in one embodiment, the serum in the medium is human AB serum at a final concentration of 5% by volume;

the final concentration of vitamin C is 30 mug/ml;

the final concentration of B18R or p65i was 0.2 μ g/ml;

the final concentration of the rapamycin is 10 ng/ml;

the final concentration of resveratrol was 10. mu.M.

The blood cleaning uses human AB serum, and the human AB serum has better effects on culturing chondrocytes and clinical application.

A kit for in vitro chondrocyte telomere elongation and proliferation culture comprises telomerase mRNA and the culture medium for in vitro chondrocyte telomere elongation and proliferation culture according to any embodiment.

In one embodiment, the base sequence of the telomerase mRNA is shown in SEQ ID No. 1.

In one embodiment, the telomerase mRNA is added to the transfection reduced serum medium.

In one embodiment, the serum-reduced medium is

I reduced serum medium (life technology, 31985070).

In one embodiment, the telomerase mRNA is present in the reduced serum medium for transfection at a concentration of 0.001-0.1. mu.g/. mu.l, preferably 0.005-0.02. mu.g/. mu.l, more preferably 0.01. mu.g/. mu.l.

In one embodiment, the telomerase mRNA is present in the reduced serum medium for transfection at a concentration of 0.01 μ g/μ l.

The culture medium for in vitro chondrocyte telomere elongation and proliferation described in any one of the embodiments above, or the kit for in vitro chondrocyte telomere elongation and proliferation described in any one of the embodiments above, is used in vitro chondrocyte telomere elongation and proliferation culture. In one embodiment, the method for culturing chondrocyte in vitro telomere elongation proliferation comprises the following steps:

the method comprises the following steps: preparing telomerase mRNA with a base sequence shown as SEQ ID No.1 in a sequence table;

step two: transfecting the telomerase mRNA into a subcultured chondrocyte in a complete culture medium, wherein the complete culture medium is the culture medium for the in vitro chondrocyte telomere elongation proliferation culture in any one embodiment;

step three: the transfected cells were continued to be cultured using the complete medium.

Wherein, in the step one, specifically:

using human genome DNA or plasmid carrying CDS region sequence of human TERT gene as template, using the sequences shown in SEQ ID No.2 and SEQ ID No.3 in the sequence table as PCR primer sequence to make PCR amplification, or making sequence complete synthesis to obtain CDS region sequence fragment of human TERT gene shown in SEQ ID No. 4;

connecting a 3 'UTR shown in SEQ ID No.5 and a 5' UTR shown in SEQ ID No.6 to two ends of the CDS region sequence fragment of the obtained human TERT gene respectively;

a poly A-containing sequence shown as SEQ ID No.7 is connected behind the 3' UTR;

linearizing the obtained DNA sequence, and transcribing the DNA sequence into mRNA in vitro;

and purifying and recovering to obtain the telomerase mRNA.

Wherein, the 5' UTR has EcoR1 enzyme cutting site (GGAATTC) at the front end, and the poly A-containing sequence shown in SEQ ID No.7 has SalI enzyme cutting site (GTCGACGCGT) at the tail end.

In which the transcription into mRNA in vitro may be carried out using an existing transcription kit, such as mMESSAGE

T7Kit (ambion AM1344), and the like.

Wherein, the purification and recovery are to obtain relatively pure mRNA by using a lithium chloride precipitation method, or further perform HPLC purification to obtain mRNA with higher purity.

Wherein, in the second step, the chondrocytes are P3-generation chondrocytes obtained by subculturing P0-generation chondrocytes separated after digesting cartilage tissue with collagenase.

The P0 generation chondrocytes are derived from a cartilage defect of a patient, and specifically, a small part of cartilage is taken from a defect part of the patient and digested by using, but not limited to, collagenase type I. The patient may be primarily targeted to elderly patients, such as patients with cartilage tissue defects above 55 years of age.

Wherein, in the second step, the density of the chondrocytes is 40% -70% when transfected. When the cell density is 40-70%, the transfection is carried out, the activity of the cell is good, and the transfection effect is ensured. Cell density refers to the proportion of the area covered by the cells to the area of the substrate.

Wherein, in the second step, the concentration of the added telomerase mRNA is 0.1-0.2 mu g/cm during transfection²。

Wherein, in the second step, the transfection comprises the following specific steps: when in transfection, a transfection reagent and a reduced serum culture medium for transfection are mixed to prepare a mixed solution A, telomerase mRNA is diluted by the corresponding reduced serum culture medium for transfection to prepare a solution B, the solution A and the solution B are mixed and incubated, the complete culture medium and the mixture of the solution A and the solution B are added into P3 generation chondrocytes, and the mixture is uniformly mixed and cultured.

Wherein the transfection reagent is

RNAImax Transfection Reagent (Life technology, 13778150) and the reduced serum medium for Transfection is

I reduced serum medium (Lifetechnology, 31985070), and transfection using the same series or similar transfection reagents.

And C, repeating the transfection method in the step II and performing one or more times of transfection on the cultured chondrocytes after transfection according to the density of the chondrocytes.

The cells cultured by the method for culturing the chondrocyte in vitro telomere extension proliferation can be used for tissue regeneration engineering, such as human tissue engineering regenerated cartilage, including collagen membranes and the chondrocytes obtained by culturing the method for culturing the chondrocyte in vitro telomere extension proliferation in any embodiment; the chondrocytes are attached to the collagen membrane.

Wherein the collagen membrane is a Bio-Gide scaffold, or similar tissue engineered collagen membrane. Collagen membrane provides a solid support for chondrocyte attachment and growth.

Wherein the chondrocyte is present at a concentration of 1X 10⁷-4×10⁷The cell suspension of individual chondrocytes/ml is prepared according to 1%10⁶-4×10⁶Cartilage cells/cm²The inoculation density is dropwise inoculated on the collagen membrane to prepare the collagen membrane.

Wherein the survival rate of the chondrocytes in the cell suspension is controlled to be not less than 80%, and the attachment rate on a collagen membrane is not less than 80%.

In one embodiment, the human tissue engineered regenerated cartilage can be transported in sterile and clean transport containers with a product expiration date of 48 hours from the end of assembly time. During transportation, the container can be packed in a packaging bag sterilized by irradiation, and the packaging bag is vacuumized and sealed. The transportation process is normal temperature transportation.

For chondrocytes, especially for old patients, no new technology is available to increase the rate of expansion, telomere length and cell activity simultaneously. By using the culture medium or the kit for in vitro chondrocyte telomere extension proliferation culture, telomerase mRNA transfection can be carried out on chondrocytes in vitro, so that telomeres in the chondrocytes obtained by subsequent culture are extended, and under the synergistic effect of the telomerase mRNA transfection and small molecular compounds such as vitamin C, B18R or p65i, rapamycin, resveratrol and the like, the proliferation and the activity of the chondrocytes, especially the chondrocytes of old patients can be remarkably improved, so that the culture medium or the kit has great significance for improving or treating cell function decline caused by aging and actually applying the cell function decline to regenerative medical treatment. The culture medium or the kit ensures that the telomeres of the chondrocytes are not continuously or frequently prolonged, has no risk of genome insertional mutagenesis and cell immortalization nodulation, greatly improves the safety and the applicability, and has better application in regenerative medical treatment.

The human tissue engineering regenerated cartilage obtained by the invention has the characteristics of obviously improving the proliferation rate and telomerase activity of chondrocytes, prolonging the telomere length and delaying the senescence of cells, and can be combined with collagen membranes and the like to form human tissue engineering regenerated cartilage products for cartilage defect treatment.

Drawings

FIG. 1 is a graph showing the cell proliferation of chondrocytes of an aged patient in example 2 after various treatments;

FIG. 2 is a graph showing the proliferation of differently treated cells of example 2 after subsequent passages;

FIG. 3 shows the results of telomere length measurement of chondrocytes of an aged patient in example 2 after various treatments;

FIG. 4 is a graph showing the results of measuring cell metabolism of chondrocytes of an aged patient in example 2 after various treatments;

FIG. 5 shows the flow detection results of chondrocyte specific marker expression of an aged patient obtained by telomerase mRNA treatment and complete culture medium culture;

FIG. 6 shows the expression of chondrocyte specific markers of an aged patient by immunofluorescence detection, after telomerase mRNA treatment and complete medium culture;

FIG. 7 shows the morphology of chondrocytes from an aged patient treated with telomerase mRNA and cultured in complete medium, detected by Alsinoblue staining.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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. Definitions of commonly used terms can be found in all molecular biology books, for example, Benjamin Lewis, Genes VIII, Oxford University Press, 2004(ISBN 0-13-145140-5). The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1 treatment of chondrocytes from an elderly patient with telomerase mRNA and complete Medium

1. Telomerase mRNA was first synthesized in vitro. The plasmid pBABE-neo-hTERT (plasmid 1774, Addgene, Cambridge, MA, USA) was PCR amplified (primers SEQ ID No.2 and SEQ ID No.3, respectively) to amplify DNA containing the CDS region of telomerase TERT (SEQ ID No. 4). The 3 'UTR (SEQ ID No.5) and 5' UTR (SEQ ID No.6) were then amplified from human genomic DNA and the full sequence synthesis of the polyA-containing sequence (SEQ ID No.7) was performed, all ligated to the DNA of the TERT CDS region using an overlapping PCR method, creating fragments: 5 'UTR-TERT CDS-3' UTR-polyA containing sequences. This fragment was then double-digested with the restriction enzymes EcoR1 and SalI and ligated into the similarly digested plasmid Pmvv 6-XL 4. And (3) verifying telomerase plasmids with correct sequences through sequencing, linearizing target gene fragments in the plasmids through enzyme digestion or PCR, and purifying and recovering the target DNA fragments by using a gel recovery kit. The linearized and purified DNA template is subjected to in vitro transcription by using an in vitro transcription kit mMESSAGE

The T7Kit (ambion AM1344) was transcribed in vitro as described in the instructions. Then the products after in vitro transcription are precipitated by lithium chloride precipitation. After completion of the precipitation, the suspension was resuspended in RNase-free water and the concentration was diluted to 1. mu.g/. mu.l. The synthesized telomerase mRNA (SEQ ID No.1) was stored at-80 ℃.

2. Preparing a complete culture medium for chondrocytes: DMEM/F12(Hyclone) + 5% human AB serum (Innovative) + 30. mu.g/ml vitamin C (sigma) + 0.2. mu.g/ml B18R (Abcam) +10ng/ml rapamycin (sigma) + 10. mu.M resveratrol (sigma). In other embodiments, the complete medium can also be prepared by using RPMI1640 basic medium, wherein B18R can also be replaced by p65i, and 5% human AB serum can also be replaced by fetal bovine serum; the content of each component is not limited to the above, for example, the content of vitamin C can be controlled to be 5-50 mug/ml, the content of B18R or p65i can be controlled to be 0.01-2 mug/ml, the content of rapamycin can be controlled to be 0.1-50ng/ml, the content of resveratrol can be controlled to be 0.1-50 muM, and the content of human AB serum can be controlled to be 1-20%; furthermore, the content of vitamin C can be controlled to be 20-40 mug/ml, the content of B18R or p65i can be controlled to be 0.1-0.5 mug/ml, the content of rapamycin can be controlled to be 1-10ng/ml, the content of resveratrol can be controlled to be 1-10 muM, and the content of human AB serum can be controlled to be 5-10%.

P0 generation chondrocytes: cartilage tissue of a male cartilage defect patient aged 76 years, which was obtained from the third hospital affiliated to Zhongshan university and was isolated after digestion with collagenase type I. The study was approved by the ethical committee of the third hospital affiliated with zhongshan university and informed consent of the patients was obtained. In other embodiments, the P0 generation chondrocytes can also be taken from a frozen stock of cells from a corresponding research institution, or the like.

4. Chondrocyte transfection: subculturing chondrocytes of P0 generation, taking second generation P2 chondrocytes which are being cultured, digesting for 5min by using 0.05% of pancreatin at 37 ℃ when the cell density is about 100%, culturing in a basal medium (DMEM/F12(Hyclone) + 5% of human AB serum (Innovative)) to stop digestion, then centrifuging for 5min at room temperature of 300g/min, and counting the resuspension of the basal medium. And (3) taking a 6-well plate, enabling the cells to be uniformly attached to the 6-well plate according to the number of the cells of 2 ten thousand per well, and changing the liquid once every other day. On day four, chondrocyte density in 6-well plates was transfected at 40-70% at this time. When transfection is performed, the amount of 3. mu.l per well is used

RNAImax transformation Reagent (Life technology, 13778150) and 97. mu.l

I serum-reduced Medium (Life technology, 31985070) was mixed to prepare mixture A, and 1. mu.g of telomerase mRNA was applied

I serum-reduced medium was diluted to 100. mu.l to prepare B, and then A and B were mixed and incubated at room temperature for 5 minutes. 1ml of a chondrocyte complete medium and 200. mu.l of the above A + B mixture were added to each well, mixed well, cultured at 37 ℃ and then subjected to cell exchange after 16 hours. On day six, re-transfection was performed according to the method on day four. When the cell density reaches nearly 100%, the cells are passaged or otherwise processed.

Example 2 detection of chondrocyte proliferation, telomerase Activity, telomere Length and cell metabolism after treatment with telomerase mRNA and complete Medium

This example sets 4 experiments, control, hTERT, Compound and hTERT + Compound, respectively, where control represents cells cultured without any treatment using basal medium (DMEM/F12(Hyclone) + 5% human AB serum (Innovative)); after the hTERT represents that the cells are transfected with telomerase mRNA, the cells are cultured by using a basic culture medium; compound represents that the cells are not transfected with telomerase mRNA and are cultured by using complete culture media added with various small molecular compounds; the hTERT + compound represents cells transfected with telomerase mRNA and cultured using complete medium. Primary chondrocytes were passage zero, and in the third passage, cells were seeded in wells of 6-well plates, with twenty thousand P3 passage chondrocytes per well.

When the cell density reached approximately 100%, the cells were digested with 0.05% pancreatin at 37 ℃ for 5 minutes, the digestion was stopped with the complete medium, centrifuged at 300g/min at room temperature for 5 minutes, the cells were resuspended in the corresponding medium, and the cells were counted. The specific results are shown in fig. 1, and it can be seen that after the telomerase mRNA is transfected into chondrocytes of an aged patient, the cell proliferation rate is greatly increased by using a complete medium containing various compounds for culture.

In the actual procedure, whether the third generation cell count after transfection or in the subsequent passages of cells, the transfection with telomerase mRNA and the culture in complete medium, the cell proliferation rate was significantly increased compared to the treatment of the other groups, as detailed in fig. 2.

When detecting the activity of telomerase, firstly, collecting the chondrocytes of the third generation, counting the cells, and taking 10⁵The individual cells were centrifuged at 200g/min for 5 minutes at room temperature, and the supernatant was removed. Using 20. mu.l lysate CHAPS Lysis Buffer (from telomerase detection kit)

Telomerase Detection Kit) cells were lysed and vortexed with a vortex shaker until the cell pellet disappeared. The samples were then diluted 1:20 to working concentration with CHAPS Lysis Buffer. According to endGranzyme detection kit

The Telomerase Detection Kit instructions were used for PCR amplification. After amplification, 20ml of 10% PAGE gel (12.75ml H) was prepared₂O, 10 XTBE buffer 0.5ml, 40% PAGE 6.25ml, 10% APS 120. mu.l, TEMED 24. mu.l), buffer 0.5 XTBE buffer, and DNA gel electrophoresis of the PCR products. Telomerase activity was detected by gel imaging using a Bio-Rad GelDocTMXR + imaging system. The specific test results are shown in fig. 3, wherein 1 represents a chondrocyte sample cultured in a basal medium without any treatment; 2 represents cells that are not transfected with telomerase mRNA, samples cultured using complete medium; 3, after the cells are transfected with telomerase mRNA, culturing the cells by using a basic culture medium; 4 represents cells transfected with telomerase mRNA, samples cultured using complete medium; 5 represents the positive control in the kit. As can be seen from FIG. 3, telomerase activity was significantly increased in chondrocytes transfected with telomerase mRNA.

When detecting Telomere Length, first, a lysate was prepared according to the instruction of the Telotaggg Telomere Length Assay kit (Roche). After the third generation chondrocytes were collected, the cells were counted, lysed by adding the above lysate at a ratio of 10,000 cells/10. mu.l, and then transferred to a 0.2ml PCR tube, and the lysed sample was placed in a constant temperature hybridization oven and rotated overnight (at least 8 hours) at 55 ℃. The next day the samples were placed in a PCR instrument and heated at 85 ℃ to inactivate the PK enzyme. The samples were placed at-20 ℃. The DNA of the sample was extracted with QIAmp DNA mini kit, the concentration of the DNA was determined using NanoDrop2000, and the DNA was diluted to 5 ng/. mu.l with RNase/DNase free water. Telomerase length detection was performed using a Bio-Rad QPCR instrument. The standard sample DNA was diluted at a concentration of 25 ng/. mu.l, 15 ng/. mu.l, 9 ng/. mu.l, 5.4 ng/. mu.l, 3.24 ng/. mu.l, and 1.94 ng/. mu.l to prepare a gradient standard sample. Telomere QPCR and genome copy number (SGC) QPCR were performed simultaneously with standard samples per plate, and a standard curve was established using CT values and corresponding standard sample concentrations. And simultaneously carrying out telomere QPCR and SGC QPCR of the sample to be detected on the same plate to obtain a corresponding CT value. And then, the telomere template concentration and the SGC template concentration of the standard sample corresponding to the sample to be detected are calculated by using the CT value and the standard curve. The telomere template concentration is proportional to the telomere length, and the telomere/SGC, T/S, value represents the average relative telomere length of the measured sample. Comparing the T/S ratio of each sample to see the change of telomere length after the cells are expanded in vitro. And calculating the T/S ratio of each sample according to the two standard curves. Changes in the T/S ratio, i.e., changes in relative telomere length, were observed. Specific results are shown in table 1, and from the results of the test in table 1, it can be seen that telomerase mRNA was transfected, and the length of telomeres was significantly increased using chondrocytes cultured in complete medium.

TABLE 1 detection of telomere length of chondrocytes from elderly patients after different treatments

When the cell metabolism was detected, the detection was carried out using alamarBlue assay kit. The specific detection result is shown in fig. 4, and it can be seen from the detection result in fig. 4 that the cell metabolism is obviously enhanced and the cell viability is increased for the cells transfected with telomerase mRNA. Complete culture medium containing various compounds is used, and under the condition of transfecting telomerase mRNA, the cell metabolism is the most vigorous, and the cell activity is the highest.

Example 3 human tissue engineered regenerative cartilage Assembly

After the chondrocytes of the aged patient are treated by telomerase mRNA and cultured by a complete culture medium containing various small molecular compounds, cell suspension is required to be prepared to be assembled and compounded on a collagen membrane bracket under the conditions of sufficient quantity and good growth state, and after the chondrocytes are completely attached to a collagen membrane, the finished products of the human tissue engineered regenerated cartilage are prepared and transported to a hospital for treatment.

AM medium (DMEM/F12(Hyclone) + 10% patient autologous serum) was first prepared. The cultured chondrocytes were subjected to cell digestion, digested for 5min with 0.05% pancreatin at 37 degrees, and the digestion was terminated by adding complete medium. After centrifugation at 300g for 5 minutes at room temperature, cells were resuspended in AM medium and counted. Centrifuging at 1500rpm for 5min, removing supernatant, and resuspending cells to obtain cell suspensionConcentration of 1X 10⁷-4×10⁷Cell seeding density of 1 × 10 per ml⁶-4×10⁶Per cm²The cell survival rate is between 80% and 100%. The sealed Bio-Gide scaffold or other similar collagen membrane scaffold is taken and cut into the required area in a biosafety cabinet by using tweezers and a disposable scalpel. Taking cell suspension with corresponding volume by using a pipette, slowly and uniformly dripping the cell suspension on the bracket from left to right and from top to bottom to ensure that the cell suspension is paved on a whole membrane without overflowing cells, putting the bracket loaded with the cells into a cell incubator, standing for 30min to avoid inclination so as to prevent the liquid level of the cells from seeping out. After the incubation is finished, cell attachment rate detection is carried out, and the cell attachment rate is required to be not less than 80%. Adding 50ml of AM culture medium into the transport container, transferring the collagen membrane scaffold into the transport container, placing the cell-loaded surface upward, covering the transport container with a cover, and keeping the product validity period within 48h from the assembly end time. The connecting part of the cover and the bottle body is wound by 3 circles by using a sealing film, the transport container is packaged by using a packaging bag sterilized by irradiation, then the vacuum-pumping sealing is carried out, and the cartilage defect treatment is carried out by transporting the cartilage defect treatment product to a hospital at normal temperature.

The following table 2 shows the quality standards of the human tissue engineered regenerative chondrocytes.

TABLE 2

As shown in FIG. 5, the aged patients obtained by telomerase mRNA treatment and complete culture medium culture have chondrocyte specific marker expression flow assay, wherein the left graph is Collagen I expression flow assay data, and the right graph is CD44 expression flow assay data. As can be seen from fig. 5, the obtained chondrocytes almost all expressed cartilage-specific surface markers, demonstrating that the cells expressed normally.

As shown in FIG. 6, immunofluorescence was performed to detect the expression of chondrocyte-specific markers of an aged patient treated with telomerase mRNA and cultured in complete medium, wherein the top panel is a Collagen I immunofluorescence image, and the bottom panel is a Collagen II immunofluorescence image. As can be seen from FIG. 6, the resulting chondrocytes specifically expressed Collagen I and Collagen II, indicating that the cells expressed normally.

As shown in FIG. 7, Alnew blue staining examined the morphology of chondrocytes from an aged patient treated with telomerase mRNA and cultured in complete medium. As can be seen from fig. 7, the obtained cells exhibited a chondrocyte-like state, and could be stained with alcian blue, indicating that the obtained chondrocyte state was normal.

Other quality standards can be effectively controlled in the assembling process of the human tissue engineered regenerative cartilage, so that the human tissue engineered regenerative cartilage assembled by the embodiment can completely meet the quality standard of the human tissue engineered regenerative chondrocytes, and can be used for transplanting the regenerative cartilage.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Zhongshan university affiliated third Hospital, Guanhao Biotechnology GmbH, Beihao Stem cell and regenerative medicine research institute GmbH

<120> culture medium for in vitro chondrocyte telomere prolonged proliferation culture, kit and application

<160>7

<170>PatentIn version 3.3

<210>1

<211>3658

<212>DNA

<213> Artificial sequence

<400>1

ggaattcaca tttgcttctg acacaactgt gttcactagc aacctcaaac agacaccatg 60

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ctgccgctgg ccacgttcgt gcggcgcctg gggccccagg gctggcggct ggtgcagcgc 180

ggggacccgg cggctttccg cgcgctggtg gcccagtgcc tggtgtgcgt gccctgggac 240

gcacggccgc cccccgccgc cccctccttc cgccaggtgt cctgcctgaa ggagctggtg 300

gcccgagtgc tgcagaggct gtgcgagcgc ggcgcgaaga acgtgctggc cttcggcttc 360

gcgctgctgg acggggcccg cgggggcccc cccgaggcct tcaccaccag cgtgcgcagc 420

tacctgccca acacggtgac cgacgcactg cgggggagcg gggcgtgggg gctgctgctg 480

cgccgcgtgg gcgacgacgt gctggttcac ctgctggcac gctgcgcgct ctttgtgctg 540

gtggctccca gctgcgccta ccaggtgtgc gggccgccgc tgtaccagct cggcgctgcc 600

actcaggccc ggcccccgcc acacgctagt ggaccccgaa ggcgtctggg atgcgaacgg 660

gcctggaacc atagcgtcag ggaggccggg gtccccctgg gcctgccagc cccgggtgcg 720

aggaggcgcg ggggcagtgc cagccgaagt ctgccgttgc ccaagaggcc caggcgtggc 780

gctgcccctg agccggagcg gacgcccgtt gggcaggggt cctgggccca cccgggcagg 840

acgcgtggac cgagtgaccg tggtttctgt gtggtgtcac ctgccagacc cgccgaagaa 900

gccacctctt tggagggtgc gctctctggc acgcgccact cccacccatc cgtgggccgc 960

cagcaccacg cgggcccccc atccacatcg cggccaccac gtccctggga cacgccttgt 1020

cccccggtgt acgccgagac caagcacttc ctctactcct caggcgacaa ggagcagctg 1080

cggccctcct tcctactcag ctctctgagg cccagcctga ctggcgctcg gaggctcgtg 1140

gagaccatct ttctgggttc caggccctgg atgccaggga ctccccgcag gttgccccgc 1200

ctgccccagc gctactggca aatgcggccc ctgtttctgg agctgcttgg gaaccacgcg 1260

cagtgcccct acggggtgct cctcaagacg cactgcccgc tgcgagctgc ggtcacccca 1320

gcagccggtg tctgtgcccg ggagaagccc cagggctctg tggcggcccc cgaggaggag 1380

gacacagacc cccgtcgcct ggtgcagctg ctccgccagc acagcagccc ctggcaggtg 1440

tacggcttcg tgcgggcctg cctgcgccgg ctggtgcccc caggcctctg gggctccagg 1500

cacaacgaac gccgcttcct caggaacacc aagaagttca tctccctggg gaagcatgcc 1560

aagctctcgc tgcaggagct gacgtggaag atgagcgtgc gggactgcgc ttggctgcgc 1620

aggagcccag gggttggctg tgttccggcc gcagagcacc gtctgcgtga ggagatcctg 1680

gccaagttcc tgcactggct gatgagtgtg tacgtcgtcg agctgctcag gtctttcttt 1740

tatgtcacgg agaccacgtt tcaaaagaac aggctctttt tctaccggaa gagtgtctgg 1800

agcaagttgc aaagcattgg aatcagacag cacttgaaga gggtgcagct gcgggagctg 1860

tcggaagcag aggtcaggca gcatcgggaa gccaggcccg ccctgctgac gtccagactc 1920

cgcttcatcc ccaagcctga cgggctgcgg ccgattgtga acatggacta cgtcgtggga 1980

gccagaacgt tccgcagaga aaagagggcc gagcgtctca cctcgagggt gaaggcactg 2040

ttcagcgtgc tcaactacga gcgggcgcgg cgccccggcc tcctgggcgc ctctgtgctg 2100

ggcctggacg atatccacag ggcctggcgc accttcgtgc tgcgtgtgcg ggcccaggac 2160

ccgccgcctg agctgtactt tgtcaaggtg gatgtgacgg gcgcgtacga caccatcccc 2220

caggacaggc tcacggaggt catcgccagc atcatcaaac cccagaacac gtactgcgtg 2280

cgtcggtatg ccgtggtcca gaaggccgcc catgggcacg tccgcaaggc cttcaagagc 2340

cacgtctcta ccttgacaga cctccagccg tacatgcgac agttcgtggc tcacctgcag 2400

gagaccagcc cgctgaggga tgccgtcgtc atcgagcaga gctcctccct gaatgaggcc 2460

agcagtggcc tcttcgacgt cttcctacgc ttcatgtgcc accacgccgt gcgcatcagg 2520

ggcaagtcct acgtccagtg ccaggggatc ccgcagggct ccatcctctc cacgctgctc 2580

tgcagcctgt gctacggcga catggagaac aagctgtttg cggggattcg gcgggacggg 2640

ctgctcctgc gtttggtgga tgatttcttg ttggtgacac ctcacctcac ccacgcgaaa 2700

accttcctca ggaccctggt ccgaggtgtc cctgagtatg gctgcgtggt gaacttgcgg 2760

aagacagtgg tgaacttccc tgtagaagac gaggccctgg gtggcacggc ttttgttcag 2820

atgccggccc acggcctatt cccctggtgc ggcctgctgc tggatacccg gaccctggag 2880

gtgcagagcg actactccag ctatgcccgg acctccatca gagccagtct caccttcaac 2940

cgcggcttca aggctgggag gaacatgcgt cgcaaactct ttggggtctt gcggctgaag 3000

tgtcacagcc tgtttctgga tttgcaggtg aacagcctcc agacggtgtg caccaacatc 3060

tacaagatcc tcctgctgca ggcgtacagg tttcacgcat gtgtgctgca gctcccattt 3120

catcagcaag tttggaagaa ccccacattt ttcctgcgcg tcatctctga cacggcctcc 3180

ctctgctact ccatcctgaa agccaagaac gcagggatgt cgctgggggc caagggcgcc 3240

gccggccctc tgccctccga ggccgtgcag tggctgtgcc accaagcatt cctgctcaag 3300

ctgactcgac accgtgtcac ctacgtgcca ctcctggggt cactcaggac agcccagacg 3360

cagctgagtc ggaagctccc ggggacgacg ctgactgccc tggaggccgc agccaacccg 3420

gcactgccct cagacttcaa gaccatcctg gactgagctc gctttcttgc tgtccaattt 3480

ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata ttatgaaggg 3540

ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa aaaaaaaaaa 3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagt cgacgcgt 3658

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<400>2

atgccgcgcg ctccccgctg 20

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<400>3

tcagtccagg atggtcttga a 21

<210>4

<211>3399

<212>DNA

<213> native sequence

<400>4

atgccgcgcg ctccccgctg ccgagccgtg cgctccctgc tgcgcagcca ctaccgcgag 60

gtgctgccgc tggccacgtt cgtgcggcgc ctggggcccc agggctggcg gctggtgcag 120

cgcggggacc cggcggcttt ccgcgcgctg gtggcccagt gcctggtgtg cgtgccctgg 180

gacgcacggc cgccccccgc cgccccctcc ttccgccagg tgtcctgcct gaaggagctg 240

gtggcccgag tgctgcagag gctgtgcgag cgcggcgcga agaacgtgct ggccttcggc 300

ttcgcgctgc tggacggggc ccgcgggggc ccccccgagg ccttcaccac cagcgtgcgc 360

agctacctgc ccaacacggt gaccgacgca ctgcggggga gcggggcgtg ggggctgctg 420

ctgcgccgcg tgggcgacga cgtgctggtt cacctgctgg cacgctgcgc gctctttgtg 480

ctggtggctc ccagctgcgc ctaccaggtg tgcgggccgc cgctgtacca gctcggcgct 540

gccactcagg cccggccccc gccacacgct agtggacccc gaaggcgtct gggatgcgaa 600

cgggcctgga accatagcgt cagggaggcc ggggtccccc tgggcctgcc agccccgggt 660

gcgaggaggc gcgggggcag tgccagccga agtctgccgt tgcccaagag gcccaggcgt 720

ggcgctgccc ctgagccgga gcggacgccc gttgggcagg ggtcctgggc ccacccgggc 780

aggacgcgtg gaccgagtga ccgtggtttc tgtgtggtgt cacctgccag acccgccgaa 840

gaagccacct ctttggaggg tgcgctctct ggcacgcgcc actcccaccc atccgtgggc 900

cgccagcacc acgcgggccc cccatccaca tcgcggccac cacgtccctg ggacacgcct 960

tgtcccccgg tgtacgccga gaccaagcac ttcctctact cctcaggcga caaggagcag 1020

ctgcggccct ccttcctact cagctctctg aggcccagcc tgactggcgc tcggaggctc 1080

gtggagacca tctttctggg ttccaggccc tggatgccag ggactccccg caggttgccc 1140

cgcctgcccc agcgctactg gcaaatgcgg cccctgtttc tggagctgct tgggaaccac 1200

gcgcagtgcc cctacggggt gctcctcaag acgcactgcc cgctgcgagc tgcggtcacc 1260

ccagcagccg gtgtctgtgc ccgggagaag ccccagggct ctgtggcggc ccccgaggag 1320

gaggacacag acccccgtcg cctggtgcag ctgctccgcc agcacagcag cccctggcag 1380

gtgtacggct tcgtgcgggc ctgcctgcgc cggctggtgc ccccaggcct ctggggctcc 1440

aggcacaacg aacgccgctt cctcaggaac accaagaagt tcatctccct ggggaagcat 1500

gccaagctct cgctgcagga gctgacgtgg aagatgagcg tgcgggactg cgcttggctg 1560

cgcaggagcc caggggttgg ctgtgttccg gccgcagagc accgtctgcg tgaggagatc 1620

ctggccaagt tcctgcactg gctgatgagt gtgtacgtcg tcgagctgct caggtctttc 1680

ttttatgtca cggagaccac gtttcaaaag aacaggctct ttttctaccg gaagagtgtc 1740

tggagcaagt tgcaaagcat tggaatcaga cagcacttga agagggtgca gctgcgggag 1800

ctgtcggaag cagaggtcag gcagcatcgg gaagccaggc ccgccctgct gacgtccaga 1860

ctccgcttca tccccaagcc tgacgggctg cggccgattg tgaacatgga ctacgtcgtg 1920

ggagccagaa cgttccgcag agaaaagagg gccgagcgtc tcacctcgag ggtgaaggca 1980

ctgttcagcg tgctcaacta cgagcgggcg cggcgccccg gcctcctggg cgcctctgtg 2040

ctgggcctgg acgatatcca cagggcctgg cgcaccttcg tgctgcgtgt gcgggcccag 2100

gacccgccgc ctgagctgta ctttgtcaag gtggatgtga cgggcgcgta cgacaccatc 2160

ccccaggaca ggctcacgga ggtcatcgcc agcatcatca aaccccagaa cacgtactgc 2220

gtgcgtcggt atgccgtggt ccagaaggcc gcccatgggc acgtccgcaa ggccttcaag 2280

agccacgtct ctaccttgac agacctccag ccgtacatgc gacagttcgt ggctcacctg 2340

caggagacca gcccgctgag ggatgccgtc gtcatcgagc agagctcctc cctgaatgag 2400

gccagcagtg gcctcttcga cgtcttccta cgcttcatgt gccaccacgc cgtgcgcatc 2460

aggggcaagt cctacgtcca gtgccagggg atcccgcagg gctccatcct ctccacgctg 2520

ctctgcagcc tgtgctacgg cgacatggag aacaagctgt ttgcggggat tcggcgggac 2580

gggctgctcc tgcgtttggt ggatgatttc ttgttggtga cacctcacct cacccacgcg 2640

aaaaccttcc tcaggaccct ggtccgaggt gtccctgagt atggctgcgt ggtgaacttg 2700

cggaagacag tggtgaactt ccctgtagaa gacgaggccc tgggtggcac ggcttttgtt 2760

cagatgccgg cccacggcct attcccctgg tgcggcctgc tgctggatac ccggaccctg 2820

gaggtgcaga gcgactactc cagctatgcc cggacctcca tcagagccag tctcaccttc 2880

aaccgcggct tcaaggctgg gaggaacatg cgtcgcaaac tctttggggt cttgcggctg 2940

aagtgtcaca gcctgtttct ggatttgcag gtgaacagcc tccagacggt gtgcaccaac 3000

atctacaaga tcctcctgct gcaggcgtac aggtttcacg catgtgtgct gcagctccca 3060

tttcatcagc aagtttggaa gaaccccaca tttttcctgc gcgtcatctc tgacacggcc 3120

tccctctgct actccatcct gaaagccaag aacgcaggga tgtcgctggg ggccaagggc 3180

gccgccggcc ctctgccctc cgaggccgtg cagtggctgt gccaccaagc attcctgctc 3240

aagctgactc gacaccgtgt cacctacgtg ccactcctgg ggtcactcag gacagcccag 3300

acgcagctga gtcggaagct cccggggacg acgctgactg ccctggaggc cgcagccaac 3360

ccggcactgc cctcagactt caagaccatc ctggactga 3399

<210>5

<211>132

<212>DNA

<213> Artificial sequence

<400>5

gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 60

taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt 120

tattttcatt gc 132

<210>6

<211>57

<212>DNA

<213> Artificial sequence

<400>6

ggaattcaca tttgcttctg acacaactgt gttcactagc aacctcaaac agacacc 57

<210>7

<211>70

<212>DNA

<213> Artificial sequence

<400>7

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

gtcgacgcgt 70

Claims (9)

1. The kit for in vitro human chondrocyte telomere prolonged proliferation culture is characterized by comprising telomerase mRNA and a culture medium for in vitro human chondrocyte telomere prolonged proliferation culture, wherein the culture medium is prepared by adding serum, vitamin C with the final concentration of 5-50 mu g/ml, B18R or p65i with the final concentration of 0.01-2 mu g/ml, rapamycin with the final concentration of 0.1-50ng/ml and resveratrol with the final concentration of 0.1-50 mu M into a DMEM/F12 or RPMI1640 basic culture medium, and the serum is human AB serum with the final volume concentration of 1-20% or bovine serum with the final volume concentration of 1-20%.

2. The kit for the in vitro human chondrocyte telomere elongation proliferation culture according to claim 1, wherein the serum in the culture medium is human AB serum with a final volume concentration of 5% to 10% or fetal bovine serum with a final volume concentration of 10% to 20%;

the final concentration of the vitamin C is 20-40 mug/ml;

the final concentration of the B18R or p65i is 0.1-0.5 μ g/ml;

the final concentration of the rapamycin is 1-10 ng/ml;

the final concentration of resveratrol is 1-10 μ M.

3. The kit for the in vitro human chondrocyte telomere elongation proliferation culture according to claim 2, wherein the serum in the culture medium is human AB serum at a final volume concentration of 5%;

the final concentration of vitamin C is 30 mug/ml;

the final concentration of B18R or p65i was 0.2 μ g/ml;

the final concentration of the rapamycin is 10 ng/ml;

the final concentration of resveratrol was 10. mu.M.

4. The kit for the in vitro human chondrocyte telomere elongation proliferation culture according to any one of claims 1 to 3, wherein the base sequence of telomerase mRNA is shown in SEQ ID No. 1.

5. The kit for the in vitro human chondrocyte telomere elongation proliferation culture according to any one of claims 1 to 3, wherein the telomerase mRNA is added to a transfection-use reduced serum medium.

6. The kit for the in vitro human chondrocyte telomere elongation proliferation culture according to claim 5, wherein the concentration of telomerase mRNA in the reduced serum medium for transfection is 0.001-0.1 μ g/μ l.

7. The kit for the in vitro human chondrocyte telomere elongation and proliferation culture according to claim 6, wherein the concentration of telomerase mRNA in the serum-reduced medium for transfection is 0.005-0.02 μ g/μ l.

8. The kit for the in vitro human chondrocyte telomere elongation proliferation culture according to claim 7, wherein the concentration of telomerase mRNA in the reduced serum medium for transfection is 0.01 μ g/μ l.

9. Use of the kit for the in vitro human chondrocyte telomere elongation proliferation culture according to any one of claims 1 to 8 in the in vitro human chondrocyte telomere elongation proliferation culture.

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