阅读说明：本技术 一种脐带干细胞制剂在制备治疗骨关节炎药物中的应用 (Application of umbilical cord stem cell preparation in preparation of osteoarthritis treatment drug ) 是由 潘若浪 张强 潘欣 张玲 戴玲华 于 2021-08-30 设计创作，主要内容包括：本公开涉及一种脐带干细胞制剂在制备治疗骨关节炎药物中的应用,所述脐带干细胞制剂中包括：脐带间充质干细胞、组织提取液和异甘草素。其中,脐带干细胞能够在一定程度上降低炎症反应,提高软骨细胞标志基因的表达,加入组织提取液、异甘草素与脐带干细胞混合使用可以有效提高脐带干细胞的修复功能,从而发挥其治疗骨关节炎的药效。(The present disclosure relates to the use of an umbilical cord stem cell preparation in the manufacture of a medicament for the treatment of osteoarthritis, the umbilical cord stem cell preparation comprising: umbilical cord mesenchymal stem cells, a tissue extract and isoliquiritigenin. The umbilical cord stem cells can reduce inflammatory reaction to a certain extent, improve the expression of chondrocyte marker genes, and effectively improve the repair function of the umbilical cord stem cells by adding tissue extract, isoliquiritigenin and the umbilical cord stem cells for mixed use, thereby exerting the drug effect of treating osteoarthritis.)

1. Use of an umbilical cord stem cell preparation in the manufacture of a medicament for the treatment of osteoarthritis, the umbilical cord stem cell preparation comprising: umbilical cord mesenchymal stem cells, a tissue extract and isoliquiritigenin.

2. The use of claim 1, wherein the umbilical cord mesenchymal stem cells are at a concentration of (0.5-5) x 10⁶one/mL.

3. The use according to claim 1, wherein the tissue extract is obtained from Wharton's jelly tissue, and the protein molecular weight of the tissue extract is 3-10KD, and the protein concentration is 0.1-0.8 mg/mL.

4. The use according to claim 1, wherein the concentration of isoliquiritigenin is 0.1-5 μ g/mL.

5. The use according to claim 1, wherein the preparation method of umbilical cord mesenchymal stem cells comprises the following steps: shearing the Wharton's jelly, placing in a serum-free complete culture medium for adherent culture, and discarding the Wharton's jelly fragments for continuous culture when the cell fusion degree is 70-90%; and after the culture is finished, digesting and dispersing the centrifugal cells, and then centrifuging, inoculating and carrying out subculture to obtain the umbilical cord mesenchymal stem cells.

6. The use of claim 5, wherein the conditions of adherent culture comprise: the culture time is 14-21 days, and the culture temperature is 37 ℃; the culture medium in the culture bottle is completely replaced every 2 to 3 days on the 7 th to 21 th days of adherent culture.

7. Use according to claim 5, wherein the conditions of subculture comprise: culturing for 3-5 days at 37 deg.C, and replacing half of subculture medium every 2-3 days.

8. The use according to claim 1, wherein the preparation method of the tissue extract comprises the following steps:

s1, crushing the umbilical cord Wharton' S jelly tissue, centrifuging and taking a tissue supernatant;

s2, adjusting the tissue supernatant to be 0.1-1mg/mL by using physiological saline;

s3, carrying out ultrafiltration treatment on the tissue supernatant with the concentration of 0.1-1mg/mL to obtain a tissue extract.

9. The use of claim 8, wherein the crushing process in step S1 comprises: the umbilical cord Wharton's jelly tissue is mixed with physiological saline and/or PBS, and then disrupted.

10. Use according to claim 9, wherein the volume ratio of umbilical cord Wharton's jelly tissue to physiological saline and/or PBS is 1: (1-5).

Technical Field

The disclosure relates to the technical field of biological medicines, in particular to application of an umbilical cord stem cell preparation in preparation of a medicine for treating osteoarthritis.

Background

Arthritis is a chronic arthritic disease with joint degeneration or aging as the pathological basis and joint swelling, pain or dysfunction as the main clinical manifestations, formerly known as osteoarthritis, degenerative osteoarthritis or hypertrophic arthritis, collectively referred to as osteoarthritis. Osteoarthritis is well-developed in the middle aged and elderly, obese patients, sports enthusiasts, or some professional athletes. With the improvement of living conditions and the development of society, the incidence of osteoarthrosis is on the trend of increasing year by year at present. It is known that more than 70% of the elderly are suffering from various joint pains, and osteoarthritis is the most common arthritis, accounting for more than 40% of all arthritis.

The current clinically used medicines can only relieve pain, but the effect is not good. With the continuous development of stem cell research, more therapeutic drugs in the field of osteoarthritis are explored, but the effect is not stable enough. Therefore, there is a need to provide a drug which has a stable effect and can effectively treat osteoarthritis.

Disclosure of Invention

The purpose of the disclosure is to provide an application of an umbilical cord stem cell preparation in preparation of a medicament for treating osteoarthritis, wherein the umbilical cord stem cell can reduce the inflammation level and improve the expression of a chondrocyte marker gene, and the tissue extract, isoliquiritigenin and the umbilical cord stem cell can be mixed for use to effectively improve the repair function of the umbilical cord stem cell, so that the medicament effect of treating osteoarthritis is exerted.

In order to achieve the above objects, the present disclosure provides use of an umbilical cord stem cell preparation for the preparation of a medicament for treating osteoarthritis, the umbilical cord stem cell preparation comprising: umbilical cord mesenchymal stem cells, a tissue extract and isoliquiritigenin.

According to the present disclosure, wherein the concentration of the umbilical cord mesenchymal stem cells is (0.5-5) × 10⁶Per mL;

according to the disclosure, the tissue extract is obtained from Wharton's jelly tissue of umbilical cord, and the molecular weight of the protein in the tissue extract is 3-10KD, and the protein concentration is 0.1-0.8 mg/mL.

According to the disclosure, the concentration of isoliquiritigenin is 0.1-5 mug/mL.

According to the present disclosure, the method for preparing umbilical cord mesenchymal stem cells comprises the following steps: shearing the Wharton's jelly, placing in a serum-free complete culture medium for adherent culture, and discarding the Wharton's jelly fragments for continuous culture when the cell fusion degree is 70-90%; after the culture is finished, digesting and dispersing the centrifugal cells, then centrifuging, inoculating and carrying out subculture to obtain umbilical cord mesenchymal stem cells; preferably, the umbilical cord mesenchymal stem cells are 3-8 generations of cells.

According to the present disclosure, wherein the conditions of adherent culture comprise: the culture time is 14-21 days, and the culture temperature is 37 ℃; completely replacing the primary culture medium in the culture bottle every 3 days on the 7 th to 21 th days of the primary culture;

according to the present disclosure, wherein the conditions of subculture comprise: culturing for 3-5 days at 37 deg.C, and replacing half of subculture medium every 2-3 days.

According to the disclosure, the preparation method of the tissue extracting solution comprises the following steps:

s1, crushing the umbilical cord Wharton' S jelly tissue, centrifuging and taking a tissue supernatant;

s2, adjusting the tissue supernatant to be 0.1-1mg/mL by using physiological saline;

s3, carrying out ultrafiltration treatment on the tissue supernatant with the concentration of 0.1-1mg/mL to obtain a tissue extract.

According to the present disclosure, the crushing process in step S1 includes: the umbilical cord Wharton's jelly tissue is mixed with physiological saline and/or PBS, and then disrupted.

According to the disclosure, the volume ratio of the umbilical cord Wharton's jelly tissue to the physiological saline and/or PBS is 1: (1-5).

The conditions of the centrifugation treatment include: centrifuging for 2-4 times at 3000-10000rpm for 10-30 min; the conditions of the ultrafiltration treatment include: centrifuging for 15-30min at 3000-.

By adopting the technical scheme, the umbilical cord stem cell preparation provided by the disclosure is applied to preparation of a medicament for treating osteoarthritis, wherein the umbilical cord stem cells can obviously reduce inflammatory reaction and improve expression of chondrocyte marker genes, and the tissue extract and the umbilical cord stem cells can be mixed for use to effectively improve the repair function of the umbilical cord stem cells, so that the drug effect of the umbilical cord stem cell preparation for treating osteoarthritis is exerted.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is HE staining results for arthritic pathological injury;

FIG. 2 is the Mankin's score for the extent of arthritis damage;

FIG. 3 shows the expression of chondrocyte marker genes.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The present disclosure provides an application of an umbilical cord stem cell preparation in the preparation of a medicament for treating osteoarthritis, the umbilical cord stem cell preparation comprises: umbilical cord mesenchymal stem cells, a tissue extract and isoliquiritigenin.

According to the disclosure, the concentration of the umbilical cord mesenchymal stem cells is (0.5-5) x 10⁶one/mL.

According to the disclosure, the tissue extract is obtained from Wharton's jelly tissue of umbilical cord, and the molecular weight of the protein in the tissue extract is 3-10KD, and the protein concentration is 0.1-0.8 mg/mL.

According to the disclosure, the concentration of isoliquiritigenin is 0.1-5 mug/mL.

According to the present disclosure, the method for preparing umbilical cord mesenchymal stem cells comprises the following steps: shearing the Wharton's jelly, placing in a serum-free complete culture medium for adherent culture, and discarding the Wharton's jelly fragments for continuous culture when the cell fusion degree is 70-90%; and after the culture is finished, digesting and dispersing the centrifugal cells, and then centrifuging, inoculating and carrying out subculture to obtain the umbilical cord mesenchymal stem cells.

The umbilical cord mesenchymal stem cells are fibroblast-like cells isolated from umbilical cord tissue, express CD73, CD90, and CD105, and do not express CD14, CD19, CD34, CD45, and HLA-DR.

According to the present disclosure, wherein the conditions of the primary culture comprise: the culture time is 14-21 days, and the culture temperature is 37 ℃; completely replacing the primary culture medium in the culture bottle every 2-3 days on the 7 th-21 th day of primary culture;

according to the present disclosure, wherein the conditions of subculture comprise: culturing for 3-5 days at 37 deg.C, and replacing half of subculture medium every 2-3 days.

According to the disclosure, the preparation method of the tissue extracting solution comprises the following steps:

s1, crushing the umbilical cord Wharton' S jelly tissue, centrifuging and taking a tissue supernatant;

s2, adjusting the tissue supernatant to be 0.1-1mg/mL by using physiological saline;

s3, carrying out ultrafiltration treatment on the tissue supernatant with the concentration of 0.1-1mg/mL to obtain a tissue extract.

Wherein, the conditions of the centrifugal treatment comprise: centrifuging for 2-4 times at 3000-10000rpm for 10-30 min;

the conditions of the ultrafiltration treatment include: centrifuging for 15-30min at 3000-.

According to the present disclosure, the crushing process in step S1 includes: the umbilical cord Wharton's jelly tissue is mixed with physiological saline and/or PBS, and then disrupted.

According to the disclosure, the volume ratio of the umbilical cord Wharton's jelly tissue to the physiological saline and/or PBS is 1: (1-5).

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

Serum-free complete media used in the present disclosure are available commercially.

Example 1

Cutting the umbilical cord into 1cm small sections, cleaning the surface by using physiological saline containing double antibodies, and cleaning blood in tissues; after the venous tube and the arterial tube in the tissue are stripped by using sterile forceps, the separated jelly is the Wharton's jelly. 1/3-2/3 of Wharton's jelly was used to isolate umbilical cord stem cells.

Preparing an umbilical cord mesenchymal stem cell preparation UCMSC: suspending umbilical cord mesenchymal stem cells in physiological saline to prepare an umbilical cord stem cell suspension with a total volume of 50 mu L, wherein the concentration of the umbilical cord mesenchymal stem cells is 2 x 10⁶one/mL.

Rat osteoarthritis model construction: injecting 100 mu L of 25mg/mL iodoacetic acid into knee joint cavities of rats by using a syringe to induce early-stage rat knee joint osteoarthritis, and completing model construction after one week.

The umbilical cord mesenchymal stem cell preparation is injected into a rat model side joint once every week for 3 times, sampling is carried out on the 7 th day after the 3 rd injection is completed, knee joint chondrocyte marker genes are detected, meanwhile, joint surface damage pathological conditions are detected through HE staining, and arthritis degree scoring (Mankin's scoring) is carried out, and the result is shown in figures 1-3.

Example 2

The preparation of umbilical cord mesenchymal stem cells and the construction process of rat model of this example are the same as those of example 1.

Preparing a tissue extracting solution: mixing the rest part of the Wharton jelly in the example 1 with the normal saline 1 according to the volume ratio of 1:5, and crushing the tissues by using a tissue homogenizer; centrifuging the crushed tissue at 3000rpm for 10min, collecting supernatant, and repeatedly centrifuging for 3 times under the condition; centrifuging at 10000rpm for 30min, collecting supernatant, and filtering with 0.22 μm filter screen to obtain the final product. Centrifuging the tissue lysate for 20min at 4 deg.C and 4000g with 10KD ultrafilter tube to obtain tissue extract 1 with molecular weight greater than 10 KD; centrifuging the filtrate of the tissue extractive solution 1 with 3KD ultrafilter tube at 4 deg.C and 4000g for 20min to obtain concentrated solution, i.e. protein with molecular weight of 3-10KD, and adjusting protein concentration to 0.5mg/mL with normal saline to obtain tissue extractive solution 2; the final filtrate is protein with molecular weight less than 3KD, and normal saline is used to adjust protein concentration to 0.5mg/mL, namely tissue extract 3.

Preparation of umbilical cord mesenchymal stem cell preparation 1: mixing umbilical cord mesenchymal stem cells, the tissue extracting solution 1 and isoliquiritigenin, adding normal saline for suspension, and preparing an umbilical cord stem cell suspension with a total volume of 50 mu L, wherein the concentration of the umbilical cord mesenchymal stem cells is 2 multiplied by 10⁶The protein concentration is 0.5mg/mL, and the isoliquiritigenin concentration is 2 mug/mL.

Injecting the umbilical cord mesenchymal stem cell preparation 1 into a rat model joint cavity; the injection is performed 3 times once a week, and on the 7 th day after the 3 rd injection is completed, the knee joint chondrocyte marker genes are sampled and detected, meanwhile, the pathological condition of joint surface damage is detected through HE staining, and arthritis degree scoring (Mankin's scoring) is performed, and the results are shown in figures 1-3.

Example 3

The preparation of umbilical cord mesenchymal stem cells and the construction process of a rat model in the embodiment are the same as those in embodiment 1; the umbilical cord tissue extract of this example was prepared in the same manner as in example 2.

Preparation of umbilical cord mesenchymal stem cell preparation 2: mixing umbilical cord mesenchymal stem cells, the tissue extract 2 and isoliquiritigenin, adding normal saline for suspension, and preparing into umbilical cord stem cell suspension with a total volume of 50 μ L, wherein the concentration of umbilical cord mesenchymal stem cells is 2 × 10⁶The protein concentration is 0.5mg/mL, and the isoliquiritigenin concentration is 2 mug/mL.

Injecting the umbilical cord mesenchymal stem cell preparation 2 into a rat model joint cavity once a week for 3 times, sampling and detecting knee joint chondrocyte marker genes on the 7 th day after the 3 rd injection, detecting the pathological condition of joint surface damage by HE staining and scoring the arthritis degree (Mankin's scoring), wherein the results are shown in figures 1-3.

Example 4

The preparation of umbilical cord mesenchymal stem cells and the construction process of a rat model in the embodiment are the same as those in embodiment 1; the umbilical cord tissue extract of this example was prepared in the same manner as in example 2.

Preparation of umbilical cord mesenchymal stem cell preparation 3: mixing umbilical cord mesenchymal stem cells, the tissue extract 3 and isoliquiritigenin, adding normal saline for suspension, and preparing into umbilical cord stem cell suspension with a total volume of 50 μ L, wherein the concentration of umbilical cord mesenchymal stem cells is 2 × 10⁶The protein concentration is 0.5mg/mL, and the isoliquiritigenin concentration is 2 mug/mL.

Injecting the umbilical cord mesenchymal stem cell preparation 3 into a rat model joint cavity once a week for 3 times, sampling and detecting knee joint chondrocyte marker genes on the 7 th day after the 3 rd injection, detecting the pathological condition of joint surface damage by HE staining and scoring the arthritis degree (Mankin's scoring), wherein the results are shown in figures 1-3.

Example 5

The preparation of umbilical cord mesenchymal stem cells and the construction process of a rat model in the embodiment are the same as those in embodiment 1; the umbilical cord tissue extract of this example was prepared in the same manner as in example 2.

Preparation of umbilical cord mesenchymal stem cell preparation 4: mixing umbilical cord mesenchymal stem cells, tissue extract 3 and isoliquiritigenin, adding normal saline to suspend, and preparing into umbilical cord stem cell suspension with total volume of 50 μ L, wherein the concentration of umbilical cord mesenchymal stem cells is 2 × 10⁶The protein concentration is 0.5mg/mL, and the isoliquiritigenin concentration is 10 mug/mL.

Injecting the umbilical cord mesenchymal stem cell preparation 4 into a rat model joint cavity once a week for 3 times, sampling and detecting knee joint chondrocyte marker genes on the 7 th day after the 3 rd injection, detecting the pathological condition of joint surface damage by HE staining and scoring the arthritis degree (Mankin's scoring), wherein the results are shown in figures 1-3.

The model group is an untreated rat model, and as is apparent from fig. 1, joint destruction of the model group is obvious and belongs to symptoms of early arthritis; the damage caused by osteoarthritis can be recovered by different treatment groups, wherein the treatment effect of the preparation 2 group is most obvious;

figure 2 scoring each group according to Mankin's scoring rule, formulation 2 group scored significantly less (× p <0.01) than the other groups, indicating that it was most effective in reducing osteoarticular damage;

in fig. 3, it is shown that the preparation 2 group can promote the expression of the chondrocyte marker genes Aggrecan and Col2a1 to be significantly better than other groups (p < 0.05;. p <0.01), indicating that the protection and the promotion of chondrocyte repair are optimal.

Comparative example 1

This comparative example was prepared in the same manner as in example 2, except that no tissue extract was added to the umbilical cord stem cell preparation in this comparative example. The results show that the umbilical cord stem cell preparation prepared by the comparative example can slightly reduce the bone joint damage and improve the expression of the chondrocyte marker gene.

Comparative example 2

This comparative example was prepared in the same manner as in example 2, except that isoliquiritigenin was not added to the umbilical cord stem cell preparation in this example. The results show that the umbilical cord stem cell preparation prepared by the comparative example can slightly reduce the bone joint damage and improve the expression of the chondrocyte marker gene.

The embodiment shows that the single umbilical cord stem cell can reduce the inflammatory level of osteoarthritis to a certain extent, improve the expression of chondrocyte marker genes and reduce the joint damage degree; the repair function of the umbilical cord stem cells cannot be improved by using the umbilical cord stem cell preparations 1, 3 and 4; the umbilical cord stem cell preparation 2, namely the umbilical cord stem cell mixed tissue extracting solution 2 (tissue extracting solution with molecular weight of 3KD-10 KD) is added with 2 mug/mL isoliquiritigenin, so that the tube arthritis score can be remarkably reduced, inflammation can be reduced, the expression of cartilage cell marker genes can be promoted, and the umbilical cord stem cell preparation 2 prepared by the method can remarkably reduce osteoarthritis damage and protect cartilage cells.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.