Application of Tripterygium wilfordii monomer in inhibiting the tumor-promoting activity of mesenchymal stem cells

Abstract

The present invention discloses the application of tripterygium wilfordii monomer in inhibiting the tumor-promoting property of mesenchymal stem cells, and relates to the field of biomedicine. The present invention treats MSCs in vitro with tripterygium wilfordii monomer, effectively inhibits the tumor-promoting property of mesenchymal stem cells MSCs, improves the safety of MSCs and its therapeutic effect on tumors, and also expands the population that can be adapted to mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a tripterygium wilfordii monomer in inhibiting the tumor-promoting activity of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are non-hematopoietic pluripotent stem cells derived from a variety of tissues including the umbilical cord, bone marrow and fat, with the ability to self-renew, proliferate and differentiate in multiple directions. Since MSCs are widely available, easy to separate, can be rapidly expanded, and have unique immunomodulatory properties, they are an ideal choice for transplantation and the treatment of immune diseases. Currently, research on MSCs in cell transplantation, tissue repair, hematopoietic support and regenerative medicine has attracted much attention. Mesenchymal stem cell preparations used in clinical transplantation therapy need to go through procedures such as tissue collection, separation and purification, primary culture, passage expansion, cryopreservation, and release testing. MSCs are the most studied and applied type of stem cells in the current field of stem cell therapy, and have increasingly attracted the favor of scientists and clinicians.

[0003] However, the current in vitro expansion of MSCs also brings some biological safety issues, especially: tumor-promoting properties. The "Guidelines for Quality Control and Preclinical Research of Stem Cell Preparations (Trial)" jointly issued by the National Health and Family Planning Commission and the State Food and Drug Administration in 2015 also pointed out: At present, it is generally believed that mesenchymal stem cells are "non-tumorigenic" or have "weak tumorigenicity", but studies have shown that MSCs have a "tumor-promoting" effect on existing tumors. Moreover, several expert consensuses believe that patients treated with MSCs need to exclude those with a history of tumors or current tumors, or those with precancerous lesions confirmed by pathological examination (Expert Consensus on Mesenchymal Stem Cells for the Treatment of New Coronavirus (2021, Beijing); Expert Consensus on Allogeneic Mesenchymal Stem Cells for the Treatment of Systemic Lupus Erythematosus (2022); Expert Consensus on Cell Transplantation for the Treatment of Severe Brain Injury and Neurological Disability in Children (2015); Expert Consensus on Standardized Treatment of Decompensated Cirrhosis with Stem Cell Transplantation (2021)). The tumor-promoting properties of MSCs have limited or hindered their application in clinical treatment to a certain extent.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide the relevant application of Tripterygium wilfordii monomer in inhibiting the tumor-promoting activity of mesenchymal stem cells.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides the use of a Tripterygium wilfordii monomer in the preparation of a product for inhibiting the tumor-promoting activity of mesenchymal stem cells.

[0008] In a second aspect, an embodiment of the present invention provides the use of Tripterygium wilfordii monomer in inhibiting the tumor-promoting activity of mesenchymal stem cells.

[0009] In the third aspect, an embodiment of the present invention provides the use of a Tripterygium wilfordii monomer in the preparation of a product for treating or assisting in the treatment of a target tumor, wherein the Tripterygium wilfordii monomer is the Tripterygium wilfordii monomer described in the preceding embodiment, and the target tumor includes any one or more of leukemia, colon tumor, and lymphoma.

[0010] The present invention has the following beneficial effects:

[0011] The present invention treats MSCs with Tripterygium wilfordii monomer in vitro, thereby effectively inhibiting the tumor-promoting property of mesenchymal stem cells MSCs, improving the therapeutic effect of MSCs on tumors, and at the same time reducing or avoiding the toxicity caused by Tripterygium wilfordii monomer entering the body, thereby expanding the population that can adapt to mesenchymal stem cells. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0013] At present, the biggest concern about the clinical application of mesenchymal stem cells is still their safety, among which the tumor-promoting property is the focus of attention in its safety. The present invention finds that the use of Tripterygium wilfordii monomer to treat MSCs in vitro can effectively inhibit the tumor-promoting property of mesenchymal stem cells MSCs, improve the safety of MSCs, and expand the applicable population of mesenchymal stem cells. It also has the characteristics of inhibiting tumors within a certain range.

[0014] On the one hand, an embodiment of the present invention provides the use of a Tripterygium wilfordii monomer in the preparation of a product for inhibiting the tumor-promoting activity of mesenchymal stem cells.

[0015] In some embodiments, the Triptergium wilfordii monomer includes any one or more of triptolide, (5R)-5-hydroxytriptolide, celastrol and celastrol propyl ester.

[0016] In some embodiments, the Tripterygium wilfordii monomer includes: triptolide and / or celastrol.

[0017] In some embodiments, the tumor-promoting property of the mesenchymal stem cells includes: the tumor-promoting property of the mesenchymal stem cells to any one or more tumors of leukemia, colon tumors and lymphomas. The colon tumors include: any one or both of benign colon tumors and malignant colon tumors (colon cancer).

[0018] In some embodiments, the source of the mesenchymal stem cells is selected from any one or more of umbilical cord, placenta, bone marrow, dental pulp, fat, amniotic membrane and endometrium.

[0019] In some embodiments, the type of product includes: a reagent or a kit.

[0020] On the other hand, an embodiment of the present invention also provides the use of Tripterygium wilfordii monomer in inhibiting the tumor-promoting activity of mesenchymal stem cells.

[0021] In some embodiments, the Tripterygium wilfordii monomer is the Tripterygium wilfordii monomer described in any of the foregoing embodiments.

[0022] In some embodiments, the source of the mesenchymal stem cells is selected from any one or more of umbilical cord, placenta, bone marrow, dental pulp, fat, amniotic membrane and endometrium.

[0023] In some embodiments, the tumor-promoting property of the mesenchymal stem cells includes: the tumor-promoting property of the mesenchymal stem cells to any one or more tumors selected from leukemia, colon tumor and lymphoma;

[0024] In some embodiments, the method of inhibiting the tumor-promoting activity of mesenchymal stem cells comprises: co-culturing Tripterygium wilfordii monomers with mesenchymal stem cells.

[0025] In some embodiments, the co-culture method can be: culturing the Tripterygium wilfordii monomer and mesenchymal stem cells in a culture medium for mesenchymal stem cells. The culture medium for mesenchymal stem cells can be selected from any existing culture medium that can be used for mesenchymal stem cell culture, for example, a serum-free culture medium.

[0026] In some embodiments, the effective concentration of the Tripterygium wilfordii monomer is 1 to 2000 ng / ml. The effective concentration can be any one of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 and 2000 ng / ml or a range between any two thereof.

[0027] In some embodiments, when the Tripterygium wilfordii monomer is triptolide or (5R)-5-hydroxytriptolide, the effective concentration of the Tripterygium wilfordii monomer is: 1 to 200 ng / ml, specifically, it can be any one of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 120, 140, 150, 160, 180, 200 ng / ml or the range between any two of them.

[0028] In some embodiments, when the Tripterygium wilfordii monomer is tripterygium wilfordii or tripterygium wilfordii propyl ester, the effective concentration of the Tripterygium wilfordii monomer is: 100-2000 ng / ml, specifically it can be any one of 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 and 2000 ng / ml or the range between any two of them.

[0029] In some embodiments, the co-culture conditions include: 1 min to 100 h. The time can specifically be any one of 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 1 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 and 100 h, or a range between any two of them.

[0030] In some embodiments, the co-culture temperature may be 36-38°C, specifically any one of 36, 36.5, 37, 37.5, 38°C or a range between any two thereof.

[0031] Optionally, when the Tripterygium wilfordii monomer is triptolide, the co-culture time is 1 min to 60 min, specifically any one of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min or a range between any two of them.

[0032] Optionally, when the Triptergium wilfordii monomer is (5R)-5-hydroxytriptolide, the co-cultivation time is 24 h to 72 h, specifically any one of 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72 h or a range between any two of them.

[0033] Optionally, when the Tripterygium wilfordii monomer is tripterygium wilfordii, the co-cultivation time is 12 h to 48 h, specifically any one of 12, 15, 20, 25, 30, 35, 40, 45, 48 h or a range between any two of them.

[0034] Optionally, when the Tripterygium wilfordii monomer is tripterygium wilfordii propyl ester, the co-cultivation time is 72 to 100 hours, specifically any one of 72, 75, 80, 85, 90, 95, 100 hours or a range between any two of them.

[0035] On the other hand, an embodiment of the present invention also provides the use of a Tripterygium wilfordii monomer in the preparation of a product for treating or assisting in the treatment of a target tumor, wherein the Tripterygium wilfordii monomer is the Tripterygium wilfordii monomer described in any of the preceding embodiments, and the target tumor includes any one or more of leukemia, colon tumor, and lymphoma.

[0036] "Treatment" as used herein includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0037] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0038] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0039] Example 1

[0040] 1. Experimental Methods

[0041] Specimen source

[0042] Human umbilical cord specimens were obtained from full-term pregnant women in the obstetrics department of Beijing Friendship Hospital. Fetal malformations, maternal congenital genetic diseases and infectious diseases were excluded, and informed consent was signed. The donors did not detect human-specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) and syphilis spirochetes, and the tests for 6-phosphate glucose dehydrogenase and alanine aminotransferase were qualified. Umbilical cord tissue was collected under sterile conditions in the operating room, washed with saline 2 to 3 times, placed in a collection bottle, and transported to the laboratory for tissue processing within 12 hours.

[0043] (1) Preparation of umbilical cord-derived MSCs:

[0044] Cut off both ends of the umbilical cord, draw out the blood from the umbilical cord blood vessels, divide the umbilical cord into small pieces of about 10 cm, soak in disinfectant for 3 minutes, and then soak in cleaning solution 2-3 times until there is no blood in the cleaning solution. Use curved forceps to tear off the two arteries and one vein inside the umbilical cord, clean the umbilical cord jelly 2-3 times, and cut into pieces of 1-2 mm 3 The tissue blocks were quickly transferred to a T25 culture flask, and 2 ml of DMEM / F12 complete medium containing 10% fetal bovine serum was added. The culture flask was transferred to a cell culture incubator at 37°C and 5% CO2 for culture. The tissue blocks were removed after 5 days, and fresh medium was replaced every 3 days. When the cells grew to about 80% confluence, 2 × 10 3 / cm 2 Density was used for subculture to obtain umbilical cord-derived MSCs.

[0045] (2) Triptolide (TP) pretreatment of MSCs:

[0046] MSCs were trypsinized, washed with PBS, and the cell concentration was adjusted to 1×10 5 After the cells adhered to the wall, the supernatant was discarded, and fresh culture medium containing 0 ng / ml, 5 ng / ml, 50 ng / ml, and 100 ng / ml TP was added and incubated for 15 minutes.

[0047] (3) In vitro study on the effect of TP-treated MSC on tumor cell proliferation

[0048] Human leukemia cell line K562 and human colon cancer cell line Colo-205 were used. The experiment set up blank group (no cell culture medium), tumor cell control group (tumor cells), MSC control group (MSC alone), MSC experimental group (MSC + tumor cells), TP + MSC experimental group (TP + MSC + tumor cells). The concentration of K562 cells was 1.0 × 10 5 / ml; Colo-205 cell concentration was 1.0×10 5 1.0×10 MSC cells / ml, 3 parallel wells were set for each concentration. 5 / ml were inoculated in 96-well culture plates, and after 24 hours 60 After Co 12.5Gy irradiation, the cells were used as co-culture layer cells. Tumor cells were inoculated 4 hours after irradiation. After 72 hours of continuous culture, the absorbance value (A) of each group of tumor cells was detected by MTT method, and the proliferation index (PI) of tumor cells was calculated. PI = (A (MSC实验组或者MSC对照组) -A (空白组) ) / (A( 肿瘤细胞对照组) -A (空白组))。The PIs of each group were statistically analyzed to compare the proliferation of tumor cells at different concentrations. A PI value within (1.00±0.20) was considered to have no effect on cell proliferation.

[0049] The results of in vitro cell experiments showed that when MSC (1.0×10 5 cells) treated with different concentrations of TP for 15 minutes was co-cultured with suspension-growing human leukemia cells K562 (1.0×10 5 cells), that is, when the corresponding cell ratio was 1:1, different concentrations of TP-treated MSC had no effect on the proliferation of K562 cells (Table 1). When MSC (1.0×10 5 cells) treated with different concentrations of TP for 15 minutes was co-cultured with adherent-growing human colon cancer cells Colo-205 (1.0×10 5 cells), that is, when the corresponding cell ratio was 1:1, different concentrations of TP-treated MSC had no effect on the proliferation of Colo-205 cells (Table 1).

[0050] Table 1. Effects of TP-treated MSC on the proliferation index PI of K562 or Colo-205 cells (x±s)

[0051] Group K562 Colo-205 MSC experimental group 1.62±0.34 1.43±0.38 5ng / ml TP+MSC experimental group 1.16±0.32 1.12±0.21 50ng / ml TP+MSC experimental group 0.96±0.22 1.02±0.19 100ng / ml TP+MSC experimental group 0.87±0.18 0.85±0.19

[0052] Note: PI≤0.80 indicates inhibition of tumor cell proliferation; PI≥1.20 indicates promotion of tumor cell proliferation; 0.80<PI<1.20 indicates no effect on tumor cell proliferation.

[0053] From the above results, it can be seen that MSC can promote the proliferation of human leukemia cells K562 and human colon cancer cells Colo-205, that is, MSC has tumor-promoting properties. After treating MSC with 5 ng / ml, 50 ng / ml, and 100 ng / ml TP, MSC lost its tumor-promoting properties.

[0054] (4) In vivo tumor-promoting experiment of TP-treated MSC on tumor-bearing mice:

[0055] One strain each of solid tumor WiDr and hematological system tumor Raji was used. The concentration of human colon cancer cells WiDr cells or human lymphoma cells Raji in the logarithmic growth phase was adjusted to 5×10 7 cells / ml with normal saline. F0-generation tumor-bearing mice were established by subcutaneous injection of 0.2 ml of WiDr cells into BALB / c nude mice and 0.2 ml of Raji cells into CB-17 SCID mice. When the tumors grew to 400 mm 3 , tumor-bearing animals with good tumor growth and health status were selected, and tumors were taken under sterile conditions and prepared into 3 mm 3The left and right tissue blocks were inoculated subcutaneously in the right axilla of the animals to prepare the BALB / c nude mouse human colon cancer cell WiDr tumor model and the CB-17SCID mouse human lymphoma cell Raji tumor model. The model animals were randomly divided into 5 groups: control group, MSC group, 5ng / ml TP+MSC group, 50ng / ml TP+MSC group, and 100ng / ml TP+MSC group, with 7 to 8 animals in each group. A blank control group was also set up for the Raji model. The experimental groups were injected with 1×10 6 MSCs / mouse, the administration volume was 0.5 ml, once every 14 days, for a total of 2 times, and the control group was given 0.5 ml of normal saline.

[0056] After the experimental endpoint, the animals in the two model experiments were euthanized and the tumor nodules were removed. The long and short diameters of the tumors were measured, and the tumor volume (TV), relative tumor volume (RTV) and relative tumor proliferation rate T / C﹪ were calculated. The calculation formula for tumor volume is: V = 1 / 2 × a × b 2 . Where a and b represent length and width, respectively. The relative tumor volume (RTV) is calculated based on the measurement results, and the calculation formula is: RTV=Vt / V0. Where V0 is the tumor volume measured when the drug is administered in different cages (i.e., d0), and Vt is the tumor volume at each measurement. Relative tumor proliferation rate T / C (%): an evaluation index for anti-tumor activity for each human cancer xenograft tumor model. The calculation formula is as follows: T / C%=TRTV / CRTV*100%. (TRTV: RTV of the treatment group; CRTV: RTV of the control group). If 140%>T / C%>40%, there is no effect; if T / C%≤40% or ≥140%, and P<0.05 after statistical processing, there is an effect.

[0057] The results show that on the 35th day in the WiDr tumor model, the Vt value of the MSC group alone was significantly higher than that of the control group (P<0.01, Table 2), and the T / C% value was >140%, indicating that MSC has a significant promoting effect on the growth of WiDr tumors. There was no statistically significant difference in the Vt values ​​of the 5ng / ml TP+MSC experimental group and the 50ng / ml TP+MSC experimental group compared with the control group (P>0.05, Table 2). The Vt value of the 100ng / ml TP+MSC experimental group was lower than that of the control group (P<0.05, Table 2), but the T / C% value was >40%.

[0058] The results showed that on the 28th day in the Raji model tumor, the Vt value of the MSC group alone was significantly higher than that of the control group (P<0.01, Table 2), and the T / C﹪ value was >140%, indicating that MSC had a significant promoting effect on the growth of Raji tumors. There was no significant difference in the Vt values ​​of the 5ng / ml TP+MSC experimental group, the 50ng / ml TP+MSC experimental group, and the 100ng / ml TP+MSC experimental group compared with the control group (P>0.05, Table 2).

[0059] Table 2. Effect of TP-treated MSCs on tumor volume Vt in WiDr or Raji models (mm 3 ,x±s)

[0060] Group WiDr (35 days) Raji (28 days) Control group 876.4±105.6 1207.2±374.8 MSC experimental group 1532.9±364.7** 2031.1±425.6** 5ng / ml TP+MSC experimental group 963.1±117.5 1324.6±417.7 50ng / ml TP+MSC experimental group 825.9±98.4 1055.0±285.2 100ng / ml TP+MSC experimental group 637.3±102.8* 899.5±157.3

[0061] Note: Compared with the control group, *P<0.05, **P<0.01.

[0062] According to the judgment criteria of the "Guidelines for Nonclinical Evaluation of Cytotoxic Anti-tumor Drugs" issued by the Drug Evaluation Center of the State Food and Drug Administration, it was determined that 5ng / ml, 50ng / ml and 100ng / ml TP treated MSCs had no promoting effect on the growth of WiDr and Raji tumors.

[0063] Example 2

[0064] (1) The human umbilical cord-derived MSCs in Example 1 were replaced with human adipose-derived MSCs. The isolation and culture procedures of adipose-derived MSCs were as follows:

[0065] The donors were excluded from congenital genetic diseases and infectious diseases, and signed informed consent. The donors were not detected with human-specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) and Treponema pallidum, and the 6-phosphate glucose dehydrogenase and alanine aminotransferase tests were qualified. The plastic surgeon performed liposuction, extracted adipose tissue, stored in a sterile collection bag or bottle, rinsed with normal saline, and cut into pieces with scissors; added 0.1% collagenase, digested at 37°C for 30 minutes; filtered through a 200-mesh cell sieve to collect nucleated cells; inoculated into complete culture medium (IMDM basal culture medium + 5% human platelet lysate + 2U / ml heparin); cultured at 37°C, 5% CO2 for 24 hours, removed non-adherent cells, and added fresh culture medium; replaced the medium in full every 2 to 3 days; when the cells grew to 80% confluence, 2×10 3 / cm 2 Subculture.

[0066] (2) LLDT-8 pretreatment of MSCs:

[0067] The pretreatment method is the same as step (2) of Example 1, except that:

[0068] The triptolide in Example 1 was replaced with (5R)-5-hydroxytriptolide (LLDT-8, C 20 H 24 O7, MW = 376.39), dissolved in DMSO as a stock solution, stored at -30 °C until use;

[0069] LLDT-8 concentrations: 5 ng / ml, 50 ng / ml, and 100 ng / ml;

[0070] The pretreatment time was 48 hours.

[0071] (3) In vitro study on the effect of LLDT-8 on tumor cell proliferation in MSCs

[0072] The experimental method is the same as step (3) in Example 1.

[0073] The results of in vitro cell experiments showed that MSCs (1.0×10 5 ) and suspension-grown human leukemia cells K562 (1.0×10 5 When the corresponding cell ratio was 1:1, MSCs treated with 5 ng / ml LLDT-8 had no effect on the proliferation of K562 cells (Table 3), but MSCs treated with 10 ng / ml LLDT-8 and 20 ng / ml LLDT-8 inhibited the proliferation of K562 cells.

[0074] MSCs (1.0×10 5 ) and adherent human colon cancer cells Colo-205 (1.0×10 5 When the corresponding cell ratio was 1:1, MSCs treated with 5 ng / ml LLDT-8 had no effect on the proliferation of Colo-205 cells, but MSCs treated with 50 ng / ml LLDT-8 and 100 ng / ml LLDT-8 inhibited the proliferation of K562 cells (Table 3).

[0075] Table 3. Effect of LLDT-8 treated MSC on proliferation index PI of K562 or Colo-205 cells (x±s)

[0076]

[0077]

[0078] Note: PI ≤ 0.80 indicates inhibition of tumor cell proliferation; PI ≥ 1.20 indicates promotion of tumor cell proliferation; 0.80 < PI < 1.20 indicates no effect on tumor cell proliferation.

[0079] It can be seen from the above results that MSC can promote the proliferation of human leukemia cell K562 and human colon cancer cell Colo-205, that is, MSC has tumor-promoting property. After MSC was treated with 5 ng / ml, 50 ng / ml and 100 ng / ml LLDT-8 for 48 hours, MSC lost its tumor-promoting property. And after MSC was treated with 50 ng / ml and 100 ng / ml LLDT-8, it had the effect of inhibiting tumor cell proliferation.

[0080] (4) In vivo tumor-promoting experiment of LLDT-8-treated MSC on tumor-bearing mice:

[0081] The experimental method was the same as step (4) in Example 1.

[0082] The results showed that: On the 35th day in the WiDr tumor model, the Vt value of the MSC-alone group was higher than that of the control group (P < 0.05, Table 4), and the T / C% value > 140%; indicating that adipose-derived MSC promoted the growth of WiDr tumors. The Vt values of the 5 ng / ml LLDT-8 + MSC experimental group, 50 ng / ml LLDT-8 + MSC experimental group and 100 ng / ml LLDT-8 + MSC experimental group were all lower than that of the control group (P < 0.05 or P < 0.01, Table 4), and the T / C% values of the 50 ng / ml LLDT-8 + MSC experimental group and 100 ng / ml LLDT-8 + MSC experimental group < 40%.

[0083] The results showed that: On the 28th day in the Raji model tumor, the Vt value of the MSC-alone group was higher than that of the control group (P < 0.05, Table 4), and the T / C% value > 140%; indicating that adipose-derived MSC significantly promoted the growth of Raji tumors. The Vt values of the 5 ng / ml LLDT-8 + MSC experimental group, 50 ng / ml LLDT-8 + MSC experimental group and 100 ng / ml LLDT-8 + MSC experimental group were all lower than that of the control group (P < 0.05 or P < 0.01, Table 4), and the T / C% values of the 50 ng / ml LLDT-8 + MSC experimental group and 100 ng / ml LLDT-8 + MSC experimental group < 40%.

[0084] Table 4. Effects of LLDT-8-treated MSC on the tumor volume Vt of WiDr or Raji model (mm 3 , x±s)

[0085]

[0086]

[0087] Note: Compared with the control group, *P<0.05.

[0088] According to the judgment criteria of the "Guidelines for Nonclinical Evaluation of Cytotoxic Anti-tumor Drugs" issued by the Drug Review Center of the State Food and Drug Administration, it was determined that MSCs treated with 5ng / ml, 50ng / ml and 100ng / ml LLDT-8 had no promoting effect on the growth of WiDr and Raji tumors, and that MSCs treated with 50ng / ml and 100ng / ml LLDT-8 had the effect of inhibiting tumor growth.

[0089] Example 3

[0090] (1) The human umbilical cord-derived MSCs in Example 1 were replaced with human bone marrow-derived MSCs. The isolation and culture procedures of bone marrow-derived MSCs were as follows:

[0091] The donors were excluded from congenital genetic diseases and infectious diseases, and signed an informed consent form. The donors were not detected with human-specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) and Treponema pallidum, and the 6-phosphate glucose dehydrogenase and alanine aminotransferase tests were qualified. Bone marrow puncture was performed by a physician who had passed the standardized residency training, and measures were taken to avoid bone marrow dilution. The bone marrow was collected into a syringe containing an anticoagulant and immediately transferred to a sterile collection bottle for storage. The human bone marrow fluid sample was diluted with sterile phosphate buffer, and the mononuclear cells were separated by density gradient centrifugation using Ficoll separation solution. Inoculated into human mesenchymal stem cell serum-free culture medium ( MSC SFM; Catalog No.: A1067501); After culturing at 37°C, 5% CO2 for 24 h, remove the non-adherent cells and add fresh culture medium; replace the medium in full every 2 to 3 days; when the cells grow to 80% confluence, add 2×10 3 / cm 2 Subculture.

[0092] (2) CEL pretreatment of MSCs:

[0093] The pretreatment method is the same as step (2) of Example 1, except that:

[0094] The triptolide in Example 1 was replaced with Celastrol (CEL);

[0095] CEL concentrations were: 15 ng / ml, 150 ng / ml, and 1500 ng / ml;

[0096] The pretreatment time was 24 hours.

[0097] (3) In vitro experiment on the effect of CEL-treated MSC on the proliferation of tumor cells

[0098] The experimental method was the same as step (3) in Example 1.

[0099] The results of in vitro cell experiments showed that when MSC treated with different concentrations of CEL for 24 hours (1.0×10 5 cells) was co-cultured with suspension-growing human leukemia cells K562 (1.0×10 5 cells), that is, when the corresponding cell ratio was 1:1, MSC treated with different concentrations of CEL had no effect on the proliferation of K562 cells (Table 5). When MSC treated with different concentrations of CEL for 24 hours (1.0×10 5 cells) was co-cultured with adherent-growing human colon cancer cells Colo-205 (1.0×10 5 cells), that is, when the corresponding cell ratio was 1:1, MSC treated with different concentrations of CEL had no effect on the proliferation of Colo-205 cells (Table 5).

[0100] Table 5. Effect of CEL-treated MSC on the proliferation index PI of K562 or Colo-205 cells (x±s)

[0101] Group K562 Colo-205 MSC experimental group 1.75±0.42 1.64±0.51 15ng / ml CEL+MSC experimental group 1.10±0.26 1.11±0.24 150ng / ml CEL+MSC experimental group 1.08±0.41 1.06±0.20 1500ng / ml CEL+MSC experimental group 0.88±0.12 0.82±0.18

[0102] Note: PI ≤ 0.80 indicates inhibition of tumor cell proliferation; PI ≥ 0.80 indicates promotion of tumor cell proliferation; 0.80 < PI < 1.20 indicates no effect on tumor cell proliferation.

[0103] It can be seen from the above results that MSC can promote the proliferation of human leukemia cells K562 and human colon cancer cells Colo-205, that is, MSC has tumor-promoting properties. After MSC was treated with 15 ng / ml, 150 ng / ml, and 1500 ng / ml CEL for 24 hours, MSC lost its tumor-promoting properties.

[0104] (4) In vivo tumor-promoting experiment of CEL-treated MSC on tumor-bearing mice:

[0105] The experimental method was the same as step (4) in Example 1.

[0106] The results showed that on the 35th day in the WiDr tumor model, the Vt value of the MSC group alone was significantly higher than that of the control group (P<0.01, Table 6), and the T / C% value was >140%, indicating that bone marrow-derived MSCs significantly promoted the growth of WiDr tumors. There was no statistically significant difference in the Vt value of the 15ng / ml CEL+MSC experimental group compared with the control group (P>0.05, Table 6). The Vt values ​​of the 150ng / ml CEL+MSC experimental group and the 1500ng / ml CEL+MSC experimental group were lower than those of the control group (P<0.05, Table 6), but the T / C% value was >40%.

[0107] The results showed that on the 28th day in the Raji model tumor, the Vt value of the MSC alone group was significantly higher than that of the control group (P<0.01, Table 6), and the T / C% value was >140%, indicating that bone marrow-derived MSCs significantly promoted the growth of Raji tumors. There was no statistically significant difference in the Vt values ​​of the 15ng / ml CEL+MSC experimental group and the 150ng / ml CEL+MSC experimental group compared with the control group (P>0.05, Table 6). The Vt value of the 1500ng / ml CEL+MSC experimental group was lower than that of the control group (P<0.05, Table 6), but the T / C% value was >40%.

[0108] Table 6. Effect of CEL-treated MSCs on tumor volume Vt in WiDr or Raji models (mm 3 ,x±s)

[0109] Group WiDr (35 days) Raji (28 days) Control group 1105.5±264.8 1148.6±225.7 MSC experimental group 1593.4±277.4** 1866.4±276.7** 15ng / ml CEL+MSC experimental group 1152.6±208.8 1227.3±293.5 150ng / ml CEL+MSC experimental group 942.4±114.7 1054.2±168.7 1500ng / ml CEL+MSC experimental group 821.5±66.2* 712.7±58.2*

[0110] Note: Compared with the control group, *P<0.05, **P<0.01.

[0111] According to the judgment criteria of the "Guidelines for Nonclinical Evaluation of Cytotoxic Anti-tumor Drugs" issued by the Drug Evaluation Center of the State Food and Drug Administration, it was determined that 15ng / ml, 150ng / ml and 1500ng / ml CEL treated MSCs had no promoting effect on the growth of WiDr and Raji tumors.

[0112] Example 4

[0113] (1) The human bone marrow-derived MSCs in Example 3 were replaced with human dental pulp-derived MSCs. The isolation and culture procedures of dental pulp-derived MSCs were as follows:

[0114] The donors were excluded from congenital genetic diseases and infectious diseases, and signed informed consent. The donors were not detected with human-specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) and Treponema pallidum, and the 6-phosphate glucose dehydrogenase and alanine aminotransferase tests were qualified. The naturally fallen deciduous teeth without tooth and pulp lesions were collected, placed in a sterile collection bottle, and the sample preservation solution was added to immerse them completely. For tooth samples, the pulp was completely removed, 0.1% collagenase was added, and digested at 37°C for 30 minutes; filtered through a 200-mesh cell sieve to collect nucleated cells. Inoculated into human mesenchymal stem cell serum-free culture medium ( MSC SFM; Catalog No.: A1067501); After culturing at 37°C, 5% CO2 for 24 h, remove the non-adherent cells and add fresh culture medium; replace the medium in full every 2 to 3 days; when the cells grow to 80% confluence, add 2×10 3 / cm 2 Subculture.

[0115] (2) MSC pretreatment with tripterygium wilfordii propyl ester:

[0116] The pretreatment method is the same as step (2) of Example 3, except that:

[0117] The tripterygium wilfordii in Example 3 was replaced with tripterygium wilfordii propyl ester, and the 28-hydroxyl group of tripterygium wilfordii was replaced with -O(CH2)2CH3 to obtain tripterygium wilfordii propyl ester;

[0118] The concentrations of tripterygium wilfordii propyl ester were: 15 ng / ml, 150 ng / ml, and 1500 ng / ml;

[0119] The pretreatment time was 96 hours.

[0120] (3) In vitro study on the effect of tripterygium wilfordii propyl ester on the proliferation of tumor cells in MSCs

[0121] The experimental method is the same as step (3) in Example 3.

[0122] The results of in vitro cell experiments showed that MSCs (1.0×10 5 ) and suspension-grown human leukemia cells K562 (1.0×10 5 When the corresponding cell ratio was 1:1, 15 ng / ml celastrol propyl ester treated MSCs had no effect on the proliferation of K562 cells, but 150 ng / ml celastrol propyl ester and 1500 ng / ml celastrol propyl ester inhibited the proliferation of K562 cells (Table 7). 5(1.0×10 adherent human colon cancer cells Colo-205 5 When co-cultured with (cells), that is, when the corresponding cell ratio is 1:1, treatment of MSC with 15 ng / ml celastrol propionate has no effect on the proliferation of Colo-205 cells, but 150 ng / ml and 1500 ng / ml celastrol propionate inhibit the proliferation of Colo-205 cells (Table 7).

[0123] Table 7. Effects of MSC treated with celastrol propionate on the proliferation index PI of K562 or Colo-205 cells (x±s)

[0124]

[0125]

[0126] Note: PI ≤ 0.80 indicates inhibition of tumor cell proliferation; PI ≥ 0.80 indicates promotion of tumor cell proliferation; 0.80 < PI < 1.20 indicates no effect on tumor cell proliferation.

[0127] It can be seen from the above results that MSC can promote the proliferation of human leukemia cells K562 and human colon cancer cells Colo-205, that is, MSC has tumor-promoting properties. After treatment of MSC with 15 ng / ml, 150 ng / ml, and 1500 ng / ml celastrol propionate for 96 hours, MSC loses its tumor-promoting properties. And treatment of MSC with 150 ng / ml and 1500 ng / ml celastrol propionate has the effect of inhibiting tumor cell proliferation.

[0128] (4) In vivo tumor-promoting experiment of MSC treated with celastrol propionate on tumor-bearing mice:

[0129] The experimental method is the same as step (4) in Example 3.

[0130] The results show that: On the 35th day in the WiDr tumor model, the Vt value of the single MSC group is higher than that of the control group (P < 0.05, Table 8), and the T / C% value > 140%; indicating that MSC has a promoting effect on the growth of WiDr tumors. The Vt values of the 150 ng / ml celastrol propionate + MSC experimental group, 1500 ng / ml celastrol propionate + MSC experimental group, and 1500 ng / ml celastrol propionate + MSC experimental group are all lower than that of the control group (P < 0.05 or P < 0.01, Table 8), and the T / C% value of the 1500 ng / ml celastrol propionate + MSC experimental group < 40%.

[0131] The results showed that on the 28th day in the Raji model tumor, the Vt value of the MSC group alone was higher than that of the control group (P<0.05, Table 8), and the T / C﹪ value was >140%, indicating that MSC had a significant promoting effect on the growth of Raji tumors. The Vt values ​​of the 150ng / ml celastrol propyl ester + MSC experimental group and the 1500ng / ml celastrol propyl ester + MSC experimental group were lower than those of the control group (P<0.05 or P<0.01, Table 8), and the T / C﹪ values ​​of the 150ng / ml celastrol propyl ester + MSC experimental group and the 1500ng / ml celastrol propyl ester + MSC experimental group were <40%.

[0132] Table 8 Effect of MSCs treated with tripterygium wilfordii propyl on Vt of WiDr or Raji tumor model (mm3, x±s)

[0133]

[0134]

[0135] Note: Compared with the control group, *P<0.05.

[0136] According to the judgment criteria of the "Guidelines for Nonclinical Evaluation of Cytotoxic Anti-tumor Drugs" issued by the Drug Review Center of the State Food and Drug Administration, it was determined that treatment of MSCs with 15ng / ml, 150ng / ml and 1500ng / ml of celastrol propyl ester had no promoting effect on the growth of WiDr and Raji tumors, and that treatment of MSCs with 150ng / ml and 1500ng / ml of celastrol propyl ester had the effect of inhibiting tumor growth.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The use of Tripterygium wilfordii monomer in the preparation of a product for inhibiting the tumor-promoting activity of mesenchymal stem cells is characterized in that: The tripterygium wilfordii monomers are: triptolide, (5R)-5-hydroxytriptolide, triptolide and triptolide propyl ester; The mesenchymal stem cells are derived from umbilical cord, bone marrow, fat or dental pulp; The application includes: triptolide inhibits the tumor-promoting effect of umbilical cord mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; and (5R)-5-hydroxytriptolide inhibits the tumor-promoting activity of adipose-derived mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; and Celastrol inhibits the tumor-promoting activity of bone marrow mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; and Celastrol propyl ester inhibits the tumor-promoting activity of dental pulp mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; The method for inhibiting the tumor-promoting activity of mesenchymal stem cells comprises: co-culturing Tripterygium wilfordii monomers with mesenchymal stem cells; Among them, when the monomer of Tripterygium wilfordii is triptolide or (5R)-5-hydroxytriptolide, the effective concentration of the monomer of Tripterygium wilfordii is: 5~200ng / ml; when the monomer of Tripterygium wilfordii is triptolide or triptolide propyl ester, the effective concentration of the monomer of Tripterygium wilfordii is: 100~2000ng / ml; When the Tripterygium wilfordii monomer is triptolide, the co-culture time is 15 minutes; When the Tripterygium wilfordii monomer is (5R)-5-hydroxytriptolide, the co-culture time is 48 hours; When the Tripterygium wilfordii monomer is tripterygium wilfordii, the co-culture time is 24 hours; When the Tripterygium wilfordii monomer is tripterygium wilfordii propyl ester, the co-culture time is 96 hours.

2. The use according to claim 1, characterized in that: The types of products include: reagents or kits.

3. The use of Tripterygium wilfordii monomer in inhibiting the tumor-promoting activity of mesenchymal stem cells, characterized in that: The tripterygium wilfordii monomers are: triptolide, (5R)-5-hydroxytriptolide, triptolide and triptolide propyl ester; The mesenchymal stem cells are derived from umbilical cord, bone marrow, fat or dental pulp; The application includes: triptolide inhibits the tumor-promoting effect of umbilical cord mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; and (5R)-5-hydroxytriptolide inhibits the tumor-promoting activity of adipose-derived mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; and Celastrol inhibits the tumor-promoting activity of bone marrow mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; and Celastrol propyl ester inhibits the tumor-promoting activity of dental pulp mesenchymal stem cells on human leukemia cells K562, human colon cancer Colo-205 cells, human colon cancer WiDr cells or human lymphoma Raji cells; The method for inhibiting the tumor-promoting activity of mesenchymal stem cells comprises: co-culturing tripterygium wilfordii monomers with mesenchymal stem cells; Among them, when the monomer of Tripterygium wilfordii is triptolide or (5R)-5-hydroxytriptolide, the effective concentration of the monomer of Tripterygium wilfordii is: 5~200ng / ml; when the monomer of Tripterygium wilfordii is triptolide or triptolide propyl ester, the effective concentration of the monomer of Tripterygium wilfordii is: 100~2000ng / ml; When the Tripterygium wilfordii monomer is triptolide, the co-culture time is 15 minutes; When the Tripterygium wilfordii monomer is (5R)-5-hydroxytriptolide, the co-culture time is 48 hours; When the Tripterygium wilfordii monomer is tripterygium wilfordii, the co-culture time is 24 hours; When the Tripterygium wilfordii monomer is celastrol propyl ester, the co-culture time is 96 hours.

4. The use according to claim 3, characterized in that: The co-cultivation temperature is 36-38°C.

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