In vitro telomere extension and proliferation culture method of chondrocytes and human tissue engineered regeneration of cartilage

Abstract

The invention discloses a method for lengthening, proliferating and culturing in-vitro telomeres of cartilage cells and human tissue engineering regenerated cartilage. The method and the human tissue engineering regenerated cartilage have the advantages that telomerase mRNA [(messenger RNA (ribonucleic acid)) transfection is carried out on the cartilage cells in an in-vitro manner, accordingly, the telomeres in cartilage cells obtained by means of culturing in follow-up procedures can be lengthened, proliferation and the viability of the cartilage cells and cartilage cells of elderly patients in particular can be obviously improved by the aid of synergistic effects of the telomerase mRNA transfection and small molecule compounds such as vitamin C, B18R or p65i, rapamycin and resveratrol in complete media, and the method and the human tissue engineering regenerated cartilage have important significance in improving or treating cell function decline due to senescence when the actually applied to regeneration medical treatment; the telomeres of the cartilage cells are not continuously or even frequently lengthened by the aid of the method for lengthening, proliferating and culturing the in-vitro telomeres of the cartilage cells, accordingly, genome insertion mutation risks or cell immortalization tumor formation risks can be prevented, the safety and the applicability can be greatly improved, and the method and the human tissue engineering regenerated cartilage can be effectively applied to the regeneration medical treatment.

Description

technical field

[0001] The invention relates to the field of cell biology, in particular to a method for chondrocyte telomere prolongation and proliferation culture in vitro and human tissue engineered regeneration cartilage. Background technique

[0002] Telomeres are protective caps at the ends of chromosomes, which are special structures composed of short, repeated non-transcribed sequences (TTAGGG) and some binding proteins, which can protect the stability of the genome and prevent the loss of genetic material at the ends of chromosomes. Cell expansion progressively affects telomere length. When the telomere is shortened to a certain extent, its protection will disappear, which will cause cell aging and apoptosis.

[0003] Telomerase is the enzyme that synthesizes telomeres and maintains their length. Telomerase consists of two parts, an RNA template subunit, the telomerase mRNA component, and a protein catalytic unit, the telomerase reverse transcriptase. Not all bod...

Examples

Embodiment 1

[0049] Example 1 Telomerase mRNA and complete medium to treat chondrocytes from elderly patients

[0050] 1. Telomerase mRNA must first be synthesized in vitro. Plasmid pBABE-neo-hTERT (plasmid 1774, Addgene, Cambridge, MA, USA) was amplified by PCR (primers were SEQ ID No.2 and SEQ ID No.3), and the TERT CDS region containing telomerase was amplified DNA (SEQ ID No.4). Then the 3'UTR (SEQ ID No.5) and 5'UTR (SEQ ID No.6) were amplified from human genomic DNA, and the polyA-containing sequence (SEQ ID No.7) was synthesized from the whole sequence, using overlapping PCR Methods All of them were connected to the DNA of the TERT CDS region to form a fragment: 5'UTR-TERT CDS-3'UTR-sequence containing polyA. Then, the fragment was double-digested with restriction endonucleases EcoR1 and SalI and ligated to the same digested plasmid Pcmv6-XL4. The telomerase plasmids with the correct sequence verified by sequencing, the target gene fragments in these plasmids are linearized by en...

Embodiment 2

[0054] Example 2 Detection of chondrocyte proliferation, telomerase activity, telomere length and cell metabolism after treatment with telomerase mRNA and complete medium

[0055] In this example, 4 groups of experiments are set up, namely control, hTERT, Compound and hTERT+compound, wherein control represents cells without any treatment, using basal medium (DMEM / F12 (Hyclone) + 5% human AB serum (Innovative)) Culture; hTERT represents cells transfected with telomerase mRNA, cultured in basal medium; Compound represents cells not transfected with telomerase mRNA, cultured in complete medium supplemented with various small molecule compounds; hTERT+compound represents cells Transfect telomerase mRNA and culture in complete medium. The primary chondrocytes were the zeroth generation, and at the third generation, the cells were planted in the wells of a 6-well plate, with 20,000 P3 generation chondrocytes per well.

[0056] When the cell density reaches nearly 100%, digest the c...

Embodiment 3

[0063] Example 3 Assembly of human tissue engineered regenerated cartilage

[0064] After the chondrocytes of elderly patients treated with telomerase mRNA and cultured in the complete medium containing various small molecular compounds are expanded to a sufficient number and in good growth state, it is necessary to prepare cell suspensions on collagen membrane scaffolds. After assembly and compounding, when the chondrocytes are completely attached to the collagen membrane, the finished human tissue engineered regenerated cartilage is made and transported to the hospital for treatment.

[0065] Firstly, AM medium (DMEM / F12 (Hyclone)+10% patient's autologous serum) was prepared. The cultured chondrocytes were taken for cell digestion, digested with 0.05% trypsin at 37°C for 5 min, and added complete medium to terminate the digestion. Centrifuge at 300g for 5 minutes at room temperature, resuspend the cells in AM medium, and count the cells. Centrifuge at 1500rpm for 5min, rem...

technical field

[0001] The invention relates to the field of cell biology, in particular to a method for chondrocyte telomere prolongation and proliferation culture in vitro and human tissue engineered regeneration cartilage. Background technique

[0002] Telomeres are protective caps at the ends of chromosomes, which are special structures composed of short, repeated non-transcribed sequences (TTAGGG) and some binding proteins, which can protect the stability of the genome and prevent the loss of genetic material at the ends of chromosomes. Cell expansion progressively affects telomere length. When the telomere is shortened to a certain extent, its protection will disappear, which will cause cell aging and apoptosis.

[0003] Telomerase is the enzyme that synthesizes telomeres and maintains their length. Telomerase consists of two parts, an RNA template subunit, the telomerase mRNA component, and a protein catalytic unit, the telomerase reverse transcriptase. Not all bod...

Examples

Embodiment 1

[0049] Example 1 Telomerase mRNA and complete medium to treat chondrocytes from elderly patients

[0050] 1. Telomerase mRNA must first be synthesized in vitro. Plasmid pBABE-neo-hTERT (plasmid 1774, Addgene, Cambridge, MA, USA) was amplified by PCR (primers were SEQ ID No.2 and SEQ ID No.3), and the TERT CDS region containing telomerase was amplified DNA (SEQ ID No.4). Then the 3'UTR (SEQ ID No.5) and 5'UTR (SEQ ID No.6) were amplified from human genomic DNA, and the polyA-containing sequence (SEQ ID No.7) was synthesized from the whole sequence, using overlapping PCR Methods All of them were connected to the DNA of the TERT CDS region to form a fragment: 5'UTR-TERT CDS-3'UTR-sequence containing polyA. Then, the fragment was double-digested with restriction endonucleases EcoR1 and SalI and ligated to the same digested plasmid Pcmv6-XL4. The telomerase plasmids with the correct sequence verified by sequencing, the target gene fragments in these plasmids are linearized by en...

Embodiment 2

[0054] Example 2 Detection of chondrocyte proliferation, telomerase activity, telomere length and cell metabolism after treatment with telomerase mRNA and complete medium

[0055] In this example, 4 groups of experiments are set up, namely control, hTERT, Compound and hTERT+compound, wherein control represents cells without any treatment, using basal medium (DMEM / F12 (Hyclone) + 5% human AB serum (Innovative)) Culture; hTERT represents cells transfected with telomerase mRNA, cultured in basal medium; Compound represents cells not transfected with telomerase mRNA, cultured in complete medium supplemented with various small molecule compounds; hTERT+compound represents cells Transfect telomerase mRNA and culture in complete medium. The primary chondrocytes were the zeroth generation, and at the third generation, the cells were planted in the wells of a 6-well plate, with 20,000 P3 generation chondrocytes per well.

[0056] When the cell density reaches nearly 100%, digest the c...

Embodiment 3

[0063] Example 3 Assembly of human tissue engineered regenerated cartilage

[0064] After the chondrocytes of elderly patients treated with telomerase mRNA and cultured in the complete medium containing various small molecular compounds are expanded to a sufficient number and in good growth state, it is necessary to prepare cell suspensions on collagen membrane scaffolds. After assembly and compounding, when the chondrocytes are completely attached to the collagen membrane, the finished human tissue engineered regenerated cartilage is made and transported to the hospital for treatment.

[0065] Firstly, AM medium (DMEM / F12 (Hyclone)+10% patient's autologous serum) was prepared. The cultured chondrocytes were taken for cell digestion, digested with 0.05% trypsin at 37°C for 5 min, and added complete medium to terminate the digestion. Centrifuge at 300g for 5 minutes at room temperature, resuspend the cells in AM medium, and count the cells. Centrifuge at 1500rpm for 5min, rem...

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