CN108969539A - Endometrial stem cells are preparing the application for preventing or treating pulmonary fibrosis medicine - Google Patents
Endometrial stem cells are preparing the application for preventing or treating pulmonary fibrosis medicine Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
Abstract
The present invention discloses endometrial stem cells and is preparing the application for preventing or treating pulmonary fibrosis medicine, belongs to field of biomedicine technology, and drug of the present invention includes endometrial stem cells, and the solvent of drug is sodium-chloride water solution.Drug of the present invention can obviously improve pulmonary fibrosis phenomenon, lower the expression of α-SMA, collagen, IL-6 in lung tissue, raise the expression of IL-10 in lung tissue；Meanwhile also lowering the expression of spleen tissue T NF- α.
Description
Technical field
The invention belongs to field of biomedicine technology more particularly to endometrial stem cells in preparation for preventing or treating lung
The application of fibrosis medicine.
Background technique
Pulmonary fibrosis is a kind of serious disease characterized by lung's permanent damage and along with high mortality, is led to
Normally due to the factors such as serious exterior trauma, spontaneous immune response or adverse drug side reaction cause the permanent damage of lung.
Studies have shown that the process of pulmonary fibrosis is roughly divided into following 3 stages: first stage, lungs are damaged or other nocuousness thorns
Swash, fibroblastic extracellular matrix (ECM) is activated cell；Second stage, the ECM of activation make cell recurring structure and
The change of phenotype generates a large amount of ECM, and mitogen original activated protein kinase intracellular and nuclear transcription factor sub-channel are activated, together
When T cell be activated, generate various cell factors；Phase III, impairment factor persistently exist, and fibroblast continues to be swashed
It is living, more ECM are generated, cell factor is continual to cause tissue inflammation and collagen to over-express, and ECM is constantly deposited, lung
Fibrosis gradually forms, and eventually leads to special hair position lung function and loses[1]。
Pulmonary fibrosis pathogenic process is a complicated pathologic process, and there are many cells and cell factor to participate in.TNF-α
(tumor necrosis factor) is an important indicator of clinical examination pulmonary fibrosis；The study found that in the lung tissue of fibrosis,
The mRNA level in-site of TNF-α is significantly higher than normal lung tissue[2], the synthesis of TNF-α and function is inhibited to can reduce or adjust pulmonary fibrosis
Process delays to enter pulmonary fibrosis acute stage.Also some researches show that IL-6 (interleukin-6) can promote the hair of pulmonary fibrosis
Raw, the expression increase of IL-6 can cause serious inflammatory reaction, cause some regions of lung tissue destructurized, cause serious
Damage, ECM are constantly deposited, and gradually form pulmonary fibrosis.And IL-10 (interleukin 10) can then inhibit pulmonary fibrosis, IL-
10 can reduce the expression of the Type I collagen generated by TGF-β stimulation, thus the pulmonary fibrosis process for preventing bleomycin from inducing.
In recent years, although with technological means progress, the research of pulmonary fibrosis mechanism and therapeutic agent obtain centainly into
Exhibition, but pathogenesis is not yet illustrated clear completely, and lacks effective treatment method.There is scholar to point out, in damage-inflammation
During three phase of disease-reparation, the imbalance of any one or more process can lead to the generation of pulmonary fibrosis, prompt us
It can prevent the formation of fibrosis by intervening above procedure[3].Currently, the primary treatment strategy of pulmonary fibrosis has anti-inflammatory, anti-fibre
Dimensionization and anti-oxidant etc., treatment means mainly have drug therapy and operative treatment etc..But drug therapy has biggish secondary work
With；And lung transplantation treats the most effective means of pulmonary fibrosis as current, due to donated organs resource shortage, rejection, sense
It the defects of dye, complication and somewhat expensive, limits its application.
There are the treatments such as document report umbilical cord mesenchymal stem cells, embryonic stem cell that can improve the lung of bleomycin induction recently
Fibrotic symptoms, but there is no report at present: endometrial stem cells are preventing or are treating the application in pulmonary fibrosis.
Bibliography
The pulmonary fibrosis mechanism such as [1] Pan Youlu, Huang Wenhai, Shen Zhengrong and Therapy study progress [J] China pharmacy
Magazine .2012,47 (23): 1873-76.
[2] Higgins DF, Kimura K, Bernhardt WM, et al.Hypoxia promotes
fibrogenesis in vivo via HIF-1stimulation of epithelial-to-mesenchymal
transition[J].J Clin Invest.2007,117(12):3810-20.
[3]Wilson MS,Wynn TA.Pulmonary fibrosis:pathogenesis,etiology and
regulation.[J].Mucosal Immunol.2009,2(2):103-21.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and endometrial stem cells are provided in preparation for preventing
Or the application for the treatment of pulmonary fibrosis medicine, the phenomenon that endometrial stem cells can obviously improve lung fiber.
To achieve the above object, the technical scheme adopted by the invention is as follows: endometrial stem cells (EMSC) are in preparation for pre-
Anti- or treatment pulmonary fibrosis medicine application.
In addition, the present invention also provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, it includes Endometriums
Stem cell.
As an improvement of the above technical solution, the density of endometrial stem cells described in pharmaceutical composition is 1.0 × 105~
5.0×106A/mL.Preferably, the density of endometrial stem cells described in pharmaceutical composition is 2.0 × 105~2.0 × 106A/
mL。
As a further improvement of the above technical scheme, the density of endometrial stem cells described in pharmaceutical composition is 1.0
×106A/mL.
As a further improvement of the above technical scheme, the solvent of pharmaceutical composition is sodium-chloride water solution.
Further improvement as above-mentioned technical proposal, the mass volume ratio of sodium chloride is in the sodium-chloride water solution
0.8%~1.0%.
Further improvement as above-mentioned technical proposal, the mass volume ratio of sodium chloride is in the sodium-chloride water solution
0.9%.
As an improvement of the above technical solution, the endometrial stem cells are taken from originally culture to the 12nd training of passage
Feeding endometrial stem cells.
In addition, the present invention also provides the preparation methods of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells are cultivated；When the density of endometrial stem cells reaches 80%~100%
When, with trypsin digestion, and collect endometrial stem cells；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution；Endometrial stem cells with
Temperature when sodium-chloride water solution mixes is 20~26 DEG C.
In addition, the present invention also provides a kind of pharmaceutical preparation comprising the pharmaceutical composition, the pharmaceutical preparation is also wrapped
Containing pharmaceutically acceptable auxiliary material, the dosage form of the pharmaceutical preparation is the solvent of intravenous injection or the solvent of intraperitoneal injection.
In the above-mentioned technical solutions, " pharmaceutically acceptable auxiliary material " includes the anti-thin of any and all physical compatibilities
Bacterium, antifungal agent, and containing a small amount of auxiliary substance that can improve shelf life or validity, such as wetting agent or emulsifier, anti-corrosion
Agent or buffer.
The beneficial effects of the present invention are: the present invention provides endometrial stem cells in preparation for preventing or treating lung fiber
The application of chemical drug object, endometrial stem cells can lower the expression of α-SMA, collagen, IL-6 in lung tissue, raise lung group
Knit the expression of middle IL-10；Meanwhile also lowering the expression of spleen tissue T NF- α；Endometrial stem cells can be obviously improved lung
Fibrosis phenomenon.
Detailed description of the invention
Fig. 1 shows the HE coloration result of lung tissue；
Fig. 2 shows the expression of α-SMA；Fig. 2A is the immunoblotting map of α-SMA, wherein β-Tublin (micro-pipe egg
It is white) as control；The expression quantity of Fig. 2 B quantitative statistics α-SMA；
The expression quantity of Fig. 3 quantitative statistics collagen, IL-6, IL-10 and TNF-α；Fig. 3 A quantitative statistics collagen's
Expression quantity；The expression quantity of Fig. 3 B quantitative statistics IL-6；The expression quantity of Fig. 3 C quantitative statistics IL-10；Fig. 3 D quantitative statistics TNF-α
Expression quantity.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiments and the drawings pair
The present invention is described further.
Embodiment 1
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes primary
The endometrial stem cells and sodium-chloride water solution of culture, the density of endometrial stem cells is 1.0 × 10 in pharmaceutical composition5A/
mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 2
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes primary
The endometrial stem cells and sodium-chloride water solution of culture, the density of endometrial stem cells is 1.0 × 10 in pharmaceutical composition6A/
mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 3
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes primary
The endometrial stem cells and sodium-chloride water solution of culture, the density of endometrial stem cells is 5.0 × 10 in pharmaceutical composition6A/
mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 4
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes passage
The endometrial stem cells and sodium-chloride water solution of 6th culture, in pharmaceutical composition the density of endometrial stem cells be 1.0 ×
105A/mL；The mass volume ratio of sodium chloride is 0.8% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 5
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes passage
The endometrial stem cells and sodium-chloride water solution of 6th culture, in pharmaceutical composition the density of endometrial stem cells be 1.0 ×
106A/mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 6
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes passage
The endometrial stem cells and sodium-chloride water solution of 6th culture, in pharmaceutical composition the density of endometrial stem cells be 5.0 ×
106A/mL；The mass volume ratio of sodium chloride is 1.0% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 7
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes passage
The endometrial stem cells and sodium-chloride water solution of tenth second incubation, the density of endometrial stem cells is 1.0 in pharmaceutical composition
×105A/mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 8
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes passage
The endometrial stem cells and sodium-chloride water solution of tenth second incubation, the density of endometrial stem cells is 2.0 in pharmaceutical composition
×105A/mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 9
The present embodiment provides a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, and pharmaceutical composition includes passage
The endometrial stem cells and sodium-chloride water solution of tenth second incubation, the density of endometrial stem cells is 2.0 in pharmaceutical composition
×106A/mL；The mass volume ratio of sodium chloride is 0.9% in sodium-chloride water solution.
The present embodiment also provides the preparation method of described pharmaceutical composition comprising following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells (EMSC) are inoculated in 10cm Tissue Culture Dish, and culture dish is set
In 37 DEG C, CO2It is cultivated in the cell incubator that concentration is 5%；When the density of endometrial stem cells reaches 80%~100%
When (preferably 90%~95%), with trypsin digestion, and endometrial stem cells are collected；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution, packing to 1mL is injected
Device is spare；Temperature when endometrial stem cells are mixed with sodium-chloride water solution is 20~26 DEG C.
Embodiment 10The therapeutic effect for the pulmonary fibrosis that pharmaceutical composition induces bleomycin
1 experimental material
1.1 experimental animal
C57BL/6 mouse: SPF grades, 6~8 week old, male；It buys in Guangdong Province's Experimental Animal Center, quality certification number are as follows:
No.44007200042765。
1.2 therapeutic combination
The pharmaceutical composition of Example 5.
1.3 main agents
Bleomycin (Bleomycin, Chinese medicines group medicine company limited liability company)；EMSC culture medium (Hangzhou Yi Wensaisheng
Object Technology Co., Ltd.)；Sodium chloride (Sigma Co., USA)；Phosphate buffer (PBS, Gibco company of the U.S.)；Dimethyl
Sulfoxide (Gibco company of the U.S.)；BCA determination of protein concentration kit (Beyotime Biotechnology)；Anti alpha-SMA antibody
(Abcam company of Britain)；Anti- β-tubulin antibody (CST company of the U.S.)；Secondary antibody (goat antirabbit, CST company of the U.S.)；
PrimerScriptTMRT reagent Kit with gDNA Eraser reverse transcription reagent box (the precious limited public affairs of biology in Dalian
Department)；Premix Ex TaqTMII (the precious biological Co., Ltd in Dalian)；Other reagents are that domestic analysis is pure.
1.4 key instrument
Superclean bench, CO2Incubator (Thermo company of the U.S.)；(Chongqing special optical instrument difficult to understand is limited for inverted microscope
Responsible company)；Low speed centrifuge, supercentrifuge (German eppendorf company)；ChemiDocTM Touch imaging
System, electrophoretic apparatus (Bio-Rad company of the U.S.)；Fluorescence quantitative PCR instrument (Applied biosystems).
2 methods
Foundation, grouping and the intervention of 2.1 pulmonary fibrosis models
6~8 week old male C57BL/6 mouse 17 is taken, wherein 12 are only given 1.5 μ g/mL bleomycins of intraperitoneal injection, often
Week 1 time, successive administration establishes pulmonary fibrosis mice model after 3 weeks；Other 5, which are only used as control group, gives intraperitoneal injection physiology salt
Water.12 be only given intraperitoneal injection 1.5 μ g/mL bleomycins mouse in first time administration 3 days after be randomly divided into model group and
EMSC group, every group 6, EMSC group gives tail vein injection pharmaceutical composition, and control group and model group give tail vein injection and go out
0.9% sodium-chloride water solution of bacterium is intervened 1 time weekly, continuous to intervene 3 weeks；And it draws materials and uses after the 21st day cervical dislocation is put to death
In following experiment.
2.2 protein immunization imprinting
2.2.1 protein extraction and quantitative
It takes lung tissue to carry out tissue grinder and extracts total protein, appropriate lysate (containing 1%PMSF) is added, is incubated for 30 points on ice
Clock；12000rpm refrigerated centrifuge 30 minutes, supernatant is transferred to new centrifuge tube；2 μ L are taken to carry out protein quantification, remaining albumen
Suitable Loading Buffer is added, as heat denatured 5 minutes in 100 DEG C after mixing, -20 DEG C are saved.
Protein quantification process is as follows: 50 times of dilution samples to be tested of lysate used, take 100 μ L that 96 porocyte culture plates are added,
According to BCA method determination of protein concentration kit specification, take A liquid, B liquid (A liquid and B liquid proportional be 1:50) in centrifuge tube respectively
In, it is made into mixed liquor after gently being blown and beaten with pipettor uniformly, every hole adds 100 μ L mixed liquors in above-mentioned well；Film is covered,
37 DEG C of water-baths are incubated for 30 minutes, detect absorbance value under microplate reader 570nm wavelength；Standard curve is drawn simultaneously according to absorbance value
Calculate the protein concentration of each sample to be tested.
2.2.2 protein electrophoresis and immunoblotting
10% separation gel is prepared, concentration glue is prepared after gelling to be separated is solid, adds to separation gel upper layer, comb is inserted into, to glue
Gently comb is extracted after body solidification；It is separately added into albumen Marker and sample in each hole 10%SDS-PAGE, 80V electrophoresis 30 divides
Zhong Hou, 120V electrophoresis 1.5 hours；In transferring film (nitrocellulose filter) 90 minutes under room temperature constant pressure 100V；Film is removed, is marked
It is closed 2 hours behind front and back sides with 5%BSA room temperature；It is cleaned 3 times, every time 5 minutes with 1 × TBS-T；According to antibody specification with 5%
BSA is diluted to working concentration, with 4 DEG C of hybridized overnights of nitrocellulose filter；It is cleaned 3 times, every time 5 minutes with 1 × TBS-T；1:
Room temperature hybridizes 1 hour after 1000 dilution secondary antibodies；It is cleaned 3 times, every time 5 minutes with 1 × TBS-T；Exposure；It scans and uses software
ImageJ (v 1.45) calculates each group protein band gray value.
2.3 real-time fluorescence quantitative PCR
2.3.1 Total RNAs extraction and reverse transcription
A small amount of tissue is taken, suitable trizol cracking is added after grinding；It places 3 minutes at room temperature, it will cracking after to be liquefied
Liquid moves in clean no enzyme microcentrifugal tube.The chloroform 0.2mL of pre-cooling is added, mixing of firmly turning upside down rapidly, ice
It is upper to stand 5 minutes, it cracks protein complexes all, is centrifuged (4 DEG C, 13000g, 15 minutes).Upper strata aqueous phase is drawn, is transferred to
Another is without 400 μ L isopropanols in enzyme centrifuge tube, are added, and mixing of firmly turning upside down rapidly, -20 DEG C stand 30 minutes, precipitating
RNA.It being centrifuged (4 DEG C, 13000g, 10 minutes), the visible a little white precipitate of tube bottom removes supernatant, 70% ethyl alcohol of 1mL is added, on
Under be mixed by inversion after wash impurity elimination, on ice place 5 minutes.It is centrifuged (4 DEG C, 7600g, 5 minutes), blots ethyl alcohol as far as possible, at room temperature
It is 5 minutes dry, it is completely transparent to RNA precipitate to remaining ethyl alcohol volatilization, 30 μ L nuclease-free waters are eventually adding, RNA is dissolved.It takes
1 μ L RNA measurement RNA concentration and purity, 260/280 ratio show that RNA mass is good between 1.8~2.0, then basis
Program in reverse transcription reagent box specification carries out the synthesis of cDNA, the template as real-time fluorescence quantitative PCR.
2.3.2 real-time fluorescence quantitative PCR
Contain in the real time reaction system of 15 μ L: the SYBR Green (5 ×) of 3 μ L, each 0.6 μ L of upstream and downstream primer
(10 μm of ol/L), 1:15 diluted cDNA 1.5 μ L, 9 μ L H2O, 0.3 μ L ROX.40 are expanded in fluorescence quantitative PCR instrument to follow
Ring, denaturation, annealing, extension required temperature, time are respectively 95 DEG C, 30s；60 DEG C, 30s；72 DEG C, 90s.Each sample repeats to survey
It is 3 times fixed.The relative quantity of mRNA indicates that average relative expression quantity passes through 2 with Ct value- △ △ CtANALYSIS OF CALCULATING；The primer sequence
As shown in table 1, β-Actin is only used as control in table 1.
Table 1
The dyeing of 2.4 lung tissue Hematoxylin-eosins (Hematoxylin-eosin, HE)
After materials, lung tissue is fixed with 10% formic acid, conventional to be dehydrated, and paraffin embedding makes wax stone, with paraffin slicing machine into
The slice of row paraffin sample, with a thickness of 3.5 μm, serial section, dyeing procedure is that roasting piece is 20 minutes dry, dimethylbenzene 2 times × 10
Minute, dehydrated alcohol 2 times × 2 minutes, 95% ethyl alcohol 1 minute, 80% ethyl alcohol 1 minute, 70% ethyl alcohol 1 minute, washing 1 minute,
It haematoxylin 8 minutes, haematoxylin 10 minutes, washes 2 times × 1 minute, 0.5% hydrochloride alcohol 10 seconds, washes 10 minutes, Yihong 2 is divided
Clock, washing 1 minute, 80%, 85%, 90% ethyl alcohol each 5 seconds, 95% ethyl alcohol 1 minute, dehydrated alcohol 2 times × 2 minutes, anhydrous second
Alcohol 3 minutes, dimethylbenzene 2 times × 2 minutes, terminate direct neutral gum mounting after dyeing, microscopy.
2.5 statistical method
Image data application Image J software carries out processing analysis, other data application statistic software SPSSs 17.0 carry out
Statistical disposition analysis, 0.05 difference of P < have statistical significance.
3 results
Influence of 3.1 pharmaceutical compositions to lung fibrosis situation
As shown in Figure 1, HE coloration result is shown, control group mice lung tissue structure is clear, has no that alveolar septum is broadening；Mould
Type group mouse lung tissue structure is destroyed seriously, and part fusion of pulmonary alveoli, alveolar septum obviously thickens, and occurs a large amount of broadbands in interstitial lung
Shape and sheet-form collagenous fiber are in diffusivity pulmonary fibrosis；EMSC group mouse lung tissue still has fibrosis to be formed, but degree of fibrosis
It is obviously reduced than model group, and has no diffuse fibrousization, diseased region is in focal distribution.
The influence that 3.2 pharmaceutical compositions express lung tissue α-SMA
As shown in Fig. 2, compared with the control group, model group mouse lung tissue Fibrosis Markers α-SMA protein expression level
It is significant to increase (P < 0.01), illustrate that mouse pulmonary fibrosis model is successfully established；Compared with model group, EMSC group mouse entirety lung
Tissue α-SMA protein expression level is remarkably decreased (P < 0.05), shows that lung fiber can be effectively relieved in pharmaceutical composition of the present invention
Change.
Influence of 3.3 pharmaceutical compositions to lung fibrosis related gene and spleen tissue T NF- alpha expression
As shown in figure 3, compared with the control group, the mRNA expression of collagen (collagen) in model group mouse lung tissue
Horizontal significant raising (P < 0.05), compared with model group, the mRNA expression of EMSC group mouse entirety expression of collagen in lung is significant
Decline (P < 0.05), shows that pulmonary fibrosis can be effectively relieved in EMSC composition.In addition, compared with model group, EMSC group mouse lung
Organize IL-6 expression is significant to lower (P < 0.05), and the significant up-regulation (P < 0.05) of IL-10 expression, while spleen tissue T NF- α
MRNA expression significantly lowers (P < 0.01), shows pharmaceutical composition of the invention in treatment pulmonary fibrosis inflammatory process hair
Wave important role；Wherein, CD3 and β-Actin is used as internal reference, and Fig. 3 is not shown.
In addition, the pharmaceutical composition of other embodiments of the invention can also obtain similar experiment effect.
4 conclusions
Pharmaceutical composition intervention is handled the pulmonary fibrosis mice model of bleomycin induction through the invention, can be significantly improved
Pulmonary fibrosis symptom: 1) being obviously improved pulmonary fibrosis phenomenon, is embodied in and lowers α-SMA albumen and collagen in lung tissue
Expression, 2) treatment pulmonary fibrosis inflammatory process play an important role, be embodied in lower lung tissue in IL-
The expression of 6 and up-regulation IL-10, and lower the expression of TNF-α in spleen tissue.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
Sequence table
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tctctctgaa ggactctggc t 21
<210> 5
<211> 18
<212> DNA
<213>artificial sequence
<400> 5
actgctaacc gactcctt 18
<210> 6
<211> 18
<212> DNA
<213>artificial sequence
<400> 6
tccactgcct tgctctta 18
<210> 7
<211> 19
<212> DNA
<213>artificial sequence
<400> 7
tgtccattcc tgagttctg 19
<210> 8
<211> 18
<212> DNA
<213>artificial sequence
<400> 8
ggaggcaaca aggtagag 18
<210> 9
<211> 20
<212> DNA
<213>artificial sequence
<400> 9
cttcatctac ggagtcatca 20
<210> 10
<211> 18
<212> DNA
<213>artificial sequence
<400> 10
gcttctgcca tcttgtct 18
<210> 11
<211> 20
<212> DNA
<213>artificial sequence
<400> 11
gcgagaagat gacccagatc 20
<210> 12
<211> 19
<212> DNA
<213>artificial sequence
<400> 12
ccagtggtac ggccagagg 19
Claims (10)
1. endometrial stem cells are preparing the application for preventing or treating pulmonary fibrosis medicine.
2. a kind of for preventing or treating the pharmaceutical composition of pulmonary fibrosis, which is characterized in that include endometrial stem cells.
3. pharmaceutical composition as claimed in claim 2, which is characterized in that endometrial stem cells described in pharmaceutical composition it is close
Degree is 1.0 × 105~5.0 × 106A/mL.
4. pharmaceutical composition as claimed in claim 3, which is characterized in that endometrial stem cells described in pharmaceutical composition it is close
Degree is 1.0 × 106A/mL.
5. pharmaceutical composition as claimed in claim 3, which is characterized in that the solvent of pharmaceutical composition is sodium-chloride water solution.
6. pharmaceutical composition as claimed in claim 5, which is characterized in that the mass body of sodium chloride in the sodium-chloride water solution
Product is than being 0.8%~1.0%.
7. pharmaceutical composition as claimed in claim 6, which is characterized in that the mass body of sodium chloride in the sodium-chloride water solution
Product is than being 0.9%.
8. the pharmaceutical composition as described in weighing and require 2, which is characterized in that the endometrial stem cells are taken from originally culture to biography
The endometrial stem cells of the tenth second incubation of generation.
9. such as the preparation method of the described in any item pharmaceutical compositions of claim 5~8, which comprises the following steps:
S1 sodium chloride) is added to the water dissolution, and carries out degerming to get sodium-chloride water solution；
S2) aseptically, endometrial stem cells are cultivated；When the density of endometrial stem cells reaches 80%~100%, use
Trypsin digestion, and collect endometrial stem cells；
S3) aseptically, the endometrial stem cells of collection are suspended from sodium-chloride water solution；Endometrial stem cells and chlorination
Temperature when sodium water solution mixes is 20~26 DEG C.
10. a kind of pharmaceutical preparation comprising the described in any item pharmaceutical compositions of such as claim 2~8, which is characterized in that described
Pharmaceutical preparation also includes pharmaceutically acceptable auxiliary material, and the dosage form of the pharmaceutical preparation is solvent or the intraperitoneal injection of intravenous injection
Solvent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810875961.5A CN108969539A (en) | 2018-08-03 | 2018-08-03 | Endometrial stem cells are preparing the application for preventing or treating pulmonary fibrosis medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810875961.5A CN108969539A (en) | 2018-08-03 | 2018-08-03 | Endometrial stem cells are preparing the application for preventing or treating pulmonary fibrosis medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108969539A true CN108969539A (en) | 2018-12-11 |
Family
ID=64554820
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810875961.5A Pending CN108969539A (en) | 2018-08-03 | 2018-08-03 | Endometrial stem cells are preparing the application for preventing or treating pulmonary fibrosis medicine |
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| CN (1) | CN108969539A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425742A (en) * | 2021-07-08 | 2021-09-24 | 苏州珈安华钰生物科技有限公司 | Application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of drugs for treating pulmonary fibrosis |
Citations (3)
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|---|---|---|---|---|
| WO2008148105A1 (en) * | 2007-05-25 | 2008-12-04 | Medistem Laboratories, Inc. | Endometrial stem cells and methods of making and using same |
| CN104666344A (en) * | 2015-02-28 | 2015-06-03 | 广州医科大学附属第一医院 | Application of MSC (mesenchymal stem cell) exosomes in preparation of pharmaceutic preparation for treating PF (pulmonary fibrosis) |
| CN106236776A (en) * | 2016-08-26 | 2016-12-21 | 杭州易文赛生物技术有限公司 | The application in preparation treatment hepatic fibrosis medicine of a kind of endometrial stem cells |
-
2018
- 2018-08-03 CN CN201810875961.5A patent/CN108969539A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008148105A1 (en) * | 2007-05-25 | 2008-12-04 | Medistem Laboratories, Inc. | Endometrial stem cells and methods of making and using same |
| CN104666344A (en) * | 2015-02-28 | 2015-06-03 | 广州医科大学附属第一医院 | Application of MSC (mesenchymal stem cell) exosomes in preparation of pharmaceutic preparation for treating PF (pulmonary fibrosis) |
| CN106236776A (en) * | 2016-08-26 | 2016-12-21 | 杭州易文赛生物技术有限公司 | The application in preparation treatment hepatic fibrosis medicine of a kind of endometrial stem cells |
Non-Patent Citations (3)
| Title |
|---|
| YIMING ZHAO，ET AL.: "Human Endometrial Regenerative Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis in Mice", 《STEM CELLS INTERNATIONAL》 * |
| 赵杨等: "肺纤维化病因表达的microRNA及其调控机制", 《武警医学》 * |
| 陈锦阳: "经血来源宫内膜干细胞的特征研究及其在肝损伤模型中的作用分析", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425742A (en) * | 2021-07-08 | 2021-09-24 | 苏州珈安华钰生物科技有限公司 | Application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of drugs for treating pulmonary fibrosis |
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