CN106333965B

CN106333965B - A kind of preparation that treating Osteoarthritis and treatment method

Info

Publication number: CN106333965B
Application number: CN201610926422.0A
Authority: CN
Inventors: 肖建辉; 张清芳
Original assignee: Affiliated Hospital of Zunyi Medical University
Current assignee: Affiliated Hospital of Zunyi Medical University
Priority date: 2016-10-31
Filing date: 2016-10-31
Publication date: 2019-04-09
Anticipated expiration: 2036-10-31
Also published as: CN106333965A

Abstract

The present invention relates to a kind of preparation for treating Osteoarthritis, said preparation uses human amnion mesenchymal stem cell and 0.01~2mg/mL molecular weight to mix for the hyaluronic acid solution of 20~2200KD, and cell density is 1 × 10⁶~1 × 10⁹A/mL, said preparation can treat Osteoarthritis and cartilage damage, and pharmaceutically acceptable carrier can also be added in said preparation.Treatment method is the hAMSCs cell preparation of every 3~5 weeks progress locally injecting equivalent in articular cavity using locally injecting 0.01~1mL of hAMSCs cell preparation to Osteoarthritis mammalian animal model, continuous 2~5 times.The cell preparation can significantly inhibit lesion inflammation, the expression for promoting glycosaminoglycan and II collagen type, repair damaged cartilage tissue, significant in efficacy.

Description

A kind of preparation that treating Osteoarthritis and treatment method

Technical field

The invention belongs to regenerative medicine and osteopathy field, in particular to a kind of preparation for treating Osteoarthritis and treatment Method.

Background technique

Osteoarthritis (osteoarthritis, OA) is a kind of common with articular cartilage retrogression pathological changes, secondary bone The chronic disease that matter hyperplasia is characterized.OA disease incidence is high, and especially mid-aged population, 50 years old or more crowd's disease incidence are more than 50%, and The disability rate of OA is up to 53%, occupies all diseases first places, and the extent of injury is more than cardiovascular and cerebrovascular disease.At present China patient there are about 1.5 hundred million, WHO speculate that global patient in 2025 will be more than 800,000,000, it has also become seriously affect the important diseases of human health, and will Annual October 12 is set to world joint buring sun.

The treatment of OA mainly includes physical therapy, drug therapy and operative treatment according to state of an illness weight.These routines Therapeutic modality, general curative effect is unsatisfactory, or even there are the series of problems such as side effect and complication at a specified future date.In recent years, being directed to Serious OA patient, the technical applications such as the transplanting of self or homogenous cartilage tissue block, Autologous Chondrocyte transplanting, there is certain treatment Effect.But the limited life span of cartilage cell, allogeneic immunogenicity, causes joint fibrosis at limited donor source The problems such as complication.Therefore, with regard to the treatment of OA, the good plan of radical cure and the key issues of osteopathy field concern be there is no at present.

The self-renewing of stem cell and the potential for being divided into multiple functions cell or even histoorgan under given conditions, Hope is brought for the treatment of injuries of tissues and organs and some difficult and complicated cases.Stem cell mainly includes embryonic stem cell, Cheng Tigan It cell and induces multi-potent stem cell, is based on ethnics Problem and Operative risk, it is dry with mesenchymal stem cell, fat mesenchymal The adult stem cells such as cell become the main seed cell resource of cellular replacement therapy.These mescenchymal stem cells all have The cell of self-renewing and Multidirectional Differentiation ability can be divided into the Various Tissues such as bone, cartilage, fat, have diving for treatment OA Power.Recently a large amount of studies have shown that human amnion mesenchymal stem cell (human amniotic mesenchymal stem Cells, hAMSCs) than adult stem cells such as mesenchymal stem cell, fat mesenchymal stem cells there is stronger amplification energy Power, differentiation capability, and be in low immunogenicity, no tumorigenesis risk and ethics limitation, and can secretory immune inhibiting factor and anti- The advantages such as the scorching factor, it is considered to be ideal seed cell (the Xiao Jianhui people amnion stem cell: regeneration of cellular replacement therapy Ideal seed cell resource Zunyi Medical College's journal 2015,38:439~449 of medicine).We grind the early period in laboratory Study carefully prompt, under certain condition, the good chondroblast differentiation capability of hAMSCs presentation (Song Xiujun, Chen Daixiong, Fang Ning, Equal human amnion mesenchymal stem cell has a potential China Tissue Engineering Study for being divided into cartilage and osteoblast, and 2007, 11:10056~10060), it is the ideal seed cell of transplantation treatment OA.However, so far there is not yet being controlled using hAMSCs transplanting Treat the report of OA.

Hyaluronic acid is a kind of by D-Glucose aldehydic acid, N- acetylaminohydroxyphenylarsonic acid D-Glucose, with -1,3 glycosidic bond and-Isosorbide-5-Nitrae sugar Glycosidic bond joins the non-protein class straight chain polymer acid mucopolysaccharide to be formed.It is also extracellular matrix principal structural component.And cell Epimatrix determines the shape of cell, influences vigor, migration, the Proliferation, Differentiation of cell, and participates in signal transmitting, and hyaluronic acid is at it In play key player, and in body performance multi-biological function normally and under pathological state.Moreover, there is study group's discovery Hyaluronic acid can be obviously promoted young rat cranium progenitor cell proliferation and induce differentiation into the ability of osteocyte, it is also considered to be structure Build the important element of cartilage tissue engineered rack material.For example, hyaluronic acid is added in chitosan stent material, it can be obvious Promote seed cell adhesive capacity and cartilage matrix generates；Hyaluronic acid is as in collagen as tissue engineering scaffold, extremely significant promotion SOX- 9, II collagen type and cartilage matrix generate.

Summary of the invention

For the deficiency of existing Osteoarthritis treatment method, the purpose of the present invention is to provide a kind of safe and efficient HAMSCs combines the arthritic preparation of hyaluronic acid transplantation treatment of bone and treatment method.

A kind of preparation for treating Osteoarthritis, said preparation the preparation method comprises the following steps: by health full term Cesarean esction fresh human placenta Passivity mechanical stripping amnion is removed after remaining bloodstain and surface mucus with 1% dual anti-D-Hank ' s liquid repeated flushing amnion is contained, Amnion is cut into 1~3 cm²Size is sub-packed in 50 mL centrifuge tubes, and addition about 2 times of volumes of amnion tissue contain 0.02% 0.05% tryptic digestive juice of EDTA-2Na, in 35~37 DEG C of constant water bath box, 150~185 rpm rotation digestion is about 30 min discard the digestive juice containing amniotic epithelial cells, rejoin fresh contain after the filtering of 300 mesh stainless steel filtering nets 0.05% tryptic digestive juice of 0.02% EDTA-2Na is repeated to digest once, be filtered again through 300 mesh stainless steel filtering nets Afterwards, digestive juice is discarded；Remaining amnion tissue cleans one with containing 1% dual anti-D-Hank ' s liquid after above-mentioned trypsin digestion It is secondary, remaining tryptic digestive juice is removed, the 0.5 mg/mL II Collagenase Type containing 0.05 mg/mL DNase I is added and disappears Change liquid, in 35~37 DEG C of constant water bath box, 150~185 rpm rotation 1~2 h of digestion, until remaining amnion tissue fragment Be digested to completely it is cotton-shaped, through 300 mesh stainless (steel) wires filter, collect cell filtrate；1500rpm, 4 DEG C of 10 min of centrifugation abandon supernatant, Cell precipitation is hAMSCs；Cell is resuspended with the low sugar DMEM containing 10% fetal calf serum, kind in T25 Tissue Culture Flask, 35~ 37 DEG C, contain 1~10% CO₂And 85~100% saturation of the air humidity constant temperature incubations, it is passed on to cell fusion degree up to 80% or more Culture；Human amnion mesenchymal stem cell and 0.01~2mg/mL hyaluronic acid solution are mixed, cell density 1 × 10⁶~1 × 10⁹A/mL is to get the cell preparation for treating Osteoarthritis.

The hAMSCs for treatment was 2~5 generations.

The molecular weight of the hyaluronic acid is 20~2200KD.

Using the method for above-mentioned preparation for treating Osteoarthritis are as follows: using locally injecting hAMSCs cell preparation 0.01~ In 1mL to the articular cavity of Osteoarthritis mammalian animal model, the hAMSCs suspension of locally injecting equivalent is carried out within every 3~5 weeks, even It is 2~5 times continuous.

HAMSCs joint hyaluronic acid for treating Osteoarthritis, be also included within wherein be added it is pharmaceutically acceptable Carrier.

The present invention is based on the superperformances of hAMSCs and hyaluronic acid, establish hAMSCs joint hyaluronic acid transplanting for the first time The preparation of Osteoarthritis is treated, and has significant curative effect, disease pain can be mitigated for many patients.

Detailed description of the invention

Fig. 1 is the gross examination of skeletal muscle (A) of normal SD rat animal articular cartilage and Osteoarthritis SD rat model, tissue disease Reason variation (B), Magnetic resonance imaging (C), glycosaminoglycan distribution (D) and II collagen type expression (E) (× 200)；

Fig. 2 is the morphological feature (× 200) before and after human amnion mesenchymal stem cell label superparamagnetic iron oxide；

Fig. 3 is the curative effect of human amnion mesenchymal stem cell and hyaluronic acid combination treatment Osteoarthritis SD rat model, (A) gross examination of skeletal muscle (× 200) after joint capsule, (B) Magnetic resonance imaging are cut；

Fig. 4 is the pathology after human amnion mesenchymal stem cell and hyaluronic acid combination treatment Osteoarthritis SD rat model Organize HE dyeing morphological feature (× 200)；

Fig. 5 is cartilage group after human amnion mesenchymal stem cell and hyaluronic acid combination treatment Osteoarthritis SD rat model The glycosaminoglycan distribution detection (× 200) knitted；

Fig. 6 is cartilage group after human amnion mesenchymal stem cell and hyaluronic acid combination treatment Osteoarthritis SD rat model The expression analysis (× 200) for the II collagen type knitted；

Fig. 7 is cartilage after human amnion mesenchymal stem cell and hyaluronic acid combination treatment Osteoarthritis SD rat model 1 expression conditions of Col2 α

Fig. 8 is blood plasma after human amnion mesenchymal stem cell and hyaluronic acid combination treatment Osteoarthritis SD rat model And in synovia inflammatory factor (A), anti-inflammatory factors and osteoprotegerin (B) expression.

Specific embodiment

The present invention is described further by following examples and attached drawing, and embodiments of the present invention are not limited thereto.

Embodiment 1: human amnion mesenchymal stem cell combine hyaluronic acid treatment Osteoarthritis preparation preparation method and Using

1, human amnion mesenchymal stem cell is separately cultured

Health full term Cesarean esction fresh human placenta passivity mechanical stripping amnion, with containing 1% dual anti-D-Hank ' s liquid (final concentration For penicillin: 100 IU/mL, streptomysin: 100 IU/mL, before use Fresh) repeated flushing amnion, remove residual bloodstain After surface mucus.Amnion is cut into 1~3 cm²Size is sub-packed in 50 mL centrifuge tubes, and about 2 times of volumes of amnion tissue are added 0.05% tryptic digestive juice containing 0.02% EDTA-2Na, in 35~37 DEG C of constant water bath box, 150~185 rpm Rotation digestion about 30 min, through 300 mesh stainless steel filtering nets filtering after, discard the digestive juice containing amniotic epithelial cells, again plus Enter fresh 0.05% tryptic digestive juice containing 0.02% EDTA-2Na, repeats to digest once, it is stainless through 300 mesh again After steel strainer filtering, digestive juice is discarded.Remaining amnion tissue, which is used, after above-mentioned trypsin digestion contains 1% dual anti-D- The cleaning of Hank ' s liquid is primary, removes remaining tryptic digestive juice, and 0.5 mg/ for containing 0.05 mg/mL DNase I is added ML II Collagenase Type digestive juice, in 35~37 DEG C of constant water bath box, 150~185 rpm rotation 1~2 h of digestion, until surplus Remaining amnion tissue fragment be digested to completely it is cotton-shaped, through 300 mesh stainless (steel) wires filter, collect cell filtrate (i.e. digestive juice), 1500 rpm, 4 DEG C of 10 min of centrifugation abandon supernatant, and cell precipitation is hAMSCs.With the low sugar DMEM weight containing 10% fetal calf serum Outstanding cell is planted in T25 Tissue Culture Flask, at 35~37 DEG C, contains 1~10% CO₂And 85~100% the saturation of the air humidity constant temperature training It supports, carries out secondary culture up to 80% or more to cell fusion degree, it is alternative in experiment to collect the 2nd~5.

2, the preparation of Osteoarthritis model

SPF grades of SD rats, age of mouse 13~14 weeks, weight: 6% chloraldurate (0.5mL/ was injected intraperitoneally in 220 ± 24g 100g weight) anesthesia.Electric shaver rejects the coat at right hind knee joint, 75% ethanol for disinfection to knee joint routine disinfection, 100 μ L of intraarticular injection (passing through ligamentum patellae) 20g/L sodium iodoacetate.Normal group gives same amount of normal saline, after modeling 2 weeks, Normal group takes 2~3 with model group at random, carries out nuclear magnetic resonance (MRI) and checks knee joint position, and in knee joint femoral and shin About 1 cm cuts joint capsule to bone up and down, carries out gross examination of skeletal muscle；HE, toluidine blue and immuning tissue are carried out to articular cartilage tissue Learn dyeing；Rat model shows as that the swelling of joint injection position, inflammation, hydrops articuli, HE staining cell be disorganized, damp line mould Paste, one or more generations (Figure 1A-E) whether the light dye of toluidine blue or mistake dye, II expression of collagen etc. in indexs.

3, superparamagnetic iron oxide (SPIO) marks hAMSCs

For convenience of therapeutic effect is checked, hAMSCs is marked using SPIO.I.e. collection is envisaged for before the hAMSCs for the treatment of (carefully Intracellular growth degrees of fusion 70~80%), tipping culture medium is added D-PBS and cleans 3 times, after exhaustion residual D-PBS, is added without FBS's The Fe that LG-DMEM culture medium is now prepared³⁺Concentration is the SPIO solution of 25 μ g/mL, is placed in 37 DEG C, 5% CO₂, 85~100% is empty It is incubated for 18 h in the incubator of gas saturated humidity, is marked, iron particle blue in visible cell (figure after prussian blue staining 2).Then, it digests, wash, collect hAMSCs, the cell suspension (cell density 1 × 10 prepared with D-PBS⁶~1 × 10⁹A/ ML).

4, animal packet combines hyaluronic acid transplantation treatment with hAMSCs

Animal is divided into Normal group (NG), model group (MG), hyaluronic acid treatment group (HA), hAMSCs treatment group (hAMSCs), hAMSCs+ hyaluronic acid combination treatment group (CG), every group 12.It is added in the cell suspension that D-PBS is prepared 0.01~2mg/mL hyaluronic acid solution mixes.The molecular weight of hyaluronic acid is 20~2200KD.HAMSCs combines hyaluronic acid For treating Osteoarthritis, it is also included within and pharmaceutically acceptable carrier is wherein added.

In position of disease, articular cavity carries out locally injecting treatment, and the 3rd~5 week after treating for the first time carries out the 2nd note Penetrate treatment.

5, hAMSCs combines hyaluronic acid transplantation treatment Osteoarthritis effect assessment

(1) gross examination of skeletal muscle, imageological examination and histopathologic change

8 weeks after treatment, solution splits knee joint position, and gross examination of skeletal muscle is shown, compares with MG group, and HA group repairs effect without obvious Fruit；HAMSCs group shows certain repairing effect；And CG group damaged cartilage repairing effect is significant (Fig. 3 A)

The prompt of Fig. 3 B Magnetic resonance imaging result, the 8th week after the treatment, model group (MG) articular cartilage tissue was mainly presented Apparent high RST, shows inflammation occur.And in articular cavity at cartilage damage tissue, HA group and hAMSCs, especially CG group Signal it is obviously on the weak side, prompt hAMSCs have moved to cartilaginous lesion site.

The prompt of pathological tissue HE coloration result, in impaired articular cartilage tissue, MG group has not observed apparent thin Born of the same parents' form, damage have reached at subchondral bone；HA group and the discovery of hAMSCs group have a small amount of cartilage cell；CG group cartilaginous tissue, cell Marshalling, damp line is clear (Fig. 4), prompts it good to repair of cartilage effect.

(2) expression of glycosaminoglycan and II Collagen Type VI

Using toluidine blue detection cartilaginous tissue glycosaminoglycan secrete situation, as a result prompt, MG group lose dye, HA group with The light dye of hAMSCs group, NG group and the dense dye of GC group show that two groups of glycosaminoglycan expression quantity are more (Fig. 5), prompt GC group to cartilage group The reparation knitted has good effect.

II collagen type is detected as shown in Fig. 6 ImmunohistochemistryResults Results, and the II collagen type of model group (MG) is negative, HA group and the expression of hAMSCs group II collagen type are lower, and normal group (NG) and combination group (CG) II collagen type are in Qiang Yang Property, prompt GC group to have good effect to the reparation of cartilaginous tissue.

(3) expression of 1 gene of cartilage Col2 α

Although the expression quantity slightly higher than MG group of HA group and hAMSCs group Col2 α 1, and the expression quantity of the Col2 α 1 of CG group is bright It is aobvious to be higher than MG group (Fig. 7), prompt HA and hAMSCs combination that there is good injury repair effect.

(4) expression of inflammatory factor, anti-inflammatory factors and OPG

The prompt of Fig. 8, compared with MG group, proinflammatory factor TNF-α, IL- in HA group, hAMSCs group and CG group blood plasma and synovia 6 and IL-1βExpression water is lowered, and wherein it is the most obvious to lower effect for CG group.On the contrary, compared with MG group, HA group, hAMSCs OPG and anti-inflammatory factors IL-4, IL-10 expression are raised in group and CG group blood plasma and synovia, wherein CG group up-regulation effect Fruit is the most obvious.Therefore, the expression of hyaluronic acid, hAMSCs and all adjustable inflammatory factor of the two combination is used alone, subtracts Subinflammation reaction promotes the secretion of OPG, and antiphlogistic effects are significant after being especially combined.

The purposes of 2. human amnion mesenchymal stem cell of embodiment and hyaluronic acid combination in other aspects

As described in the above results, human amnion mesenchymal stem cell and hyaluronic acid combination can be remarkably reinforced and cartilaginous tissue The expression of closely related glycosaminoglycan, II collagen type, and be conducive to human amnion mesenchymal stem cell in bone joint Scorching model diseased region survival is colonized and is divided into new cartilage cell, there is good preventive and therapeutic effect to cartilage damage.And And human amnion mesenchymal stem cell and hyaluronic acid combination are not also immunized for xenogenesis SD rat bone arthritis treatment Rejection.

Embodiment 3. mixes human amnion mesenchymal stem cell and 0.01~2mg/mL hyaluronic acid solution, cell density 1 ×10⁶~1 × 10⁹A/mL can be used as the cell preparation for the treatment of Osteoarthritis and cartilage damage for injection.It can also be at this Pharmaceutically acceptable carrier is added in preparation, such as promotees the growth factor of repair of cartilage.

Claims

1. a kind of preparation for treating Osteoarthritis, it is characterised in that: the preparation the preparation method comprises the following steps: health full term is cutd open palace Produce fresh human placenta passivity mechanical stripping amnion, with contain 1% dual anti-D-Hank ' s liquid repeated flushing amnion, remove residual bloodstain and After surface mucus, amnion is cut into 1~3 cm²Size is sub-packed in 50 mL centrifuge tubes, addition about 2 times of volumes of amnion tissue 0.05% tryptic digestive juice containing 0.02% EDTA-2Na, in 35~37 DEG C of constant water bath box, 150~185 rpm rotation Turn about 30 min of digestion, after the filtering of 300 mesh stainless steel filtering nets, discards the digestive juice containing amniotic epithelial cells, rejoin Fresh 0.05% tryptic digestive juice containing 0.02% EDTA-2Na repeats to digest once, again through 300 mesh stainless steels After strainer filtering, digestive juice is discarded；Remaining amnion tissue, which is used, after above-mentioned trypsin digestion contains 1% dual anti-D-Hank ' s Liquid cleaning is primary, removes remaining tryptic digestive juice, and the 0.5 mg/mL II type for containing 0.05 mg/mL DNase I is added Collagenase digesting liquid, in 35~37 DEG C of constant water bath box, 150~185 rpm rotation 1~2 h of digestion, until remaining amnion Fragment of tissue be digested to completely it is cotton-shaped, through 300 mesh stainless (steel) wires filter, collect cell filtrate；1500rpm, 4 DEG C of centrifugations 10 Min abandons supernatant, and cell precipitation is hAMSCs；Cell is resuspended with the low sugar DMEM containing 10% fetal calf serum, kind is trained in T25 cell Bottle is supported, at 35~37 DEG C, contains 1~10% CO₂And 85~100% saturation of the air humidity constant temperature incubations, to cell fusion degree up to 80% It is above to carry out secondary culture；HAMSCs and 0.01~2mg/mL molecular weight hyaluronic acid solution for being 20~2200KD are mixed, Cell density 1 × 10⁶~1 × 10⁹A/mL is to get the cell preparation for treating Osteoarthritis.

2. treating the preparation of Osteoarthritis according to claim 1, it is characterised in that: the hAMSCs was 2~5 generations.