CN116370504A - Application of umbilical cord blood stem cells in preparation of preparation for treating respiratory diseases - Google Patents
Application of umbilical cord blood stem cells in preparation of preparation for treating respiratory diseases Download PDFInfo
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- CN116370504A CN116370504A CN202310390453.9A CN202310390453A CN116370504A CN 116370504 A CN116370504 A CN 116370504A CN 202310390453 A CN202310390453 A CN 202310390453A CN 116370504 A CN116370504 A CN 116370504A
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Abstract
The invention provides an application of umbilical cord blood stem cells in preparing a preparation for treating respiratory diseases, and belongs to the technical field of biology. The preparation method of the cord blood hematopoietic stem cells comprises the step of adding stem cell separating liquid into the cord blood, wherein the stem cell separating liquid comprises, by weight, 0.1-0.5 part of tea saponin, 2-10 parts of hydroxymethyl cellulose, 4-8 parts of polysucrose and 0.1-1 part of ammonium chloride. The research result of the invention shows that the stem cell treatment is effective for respiratory diseases, and the new application of the cord blood hematopoietic stem cell provided by the invention has better application prospect for treating respiratory diseases. The umbilical cord blood mononuclear cell preparation used in the invention is used for treating respiratory diseases, and does not need blood matching type and HLA matching type.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of umbilical cord blood stem cells in preparation of a preparation for treating respiratory diseases.
Background
Respiratory diseases mainly include asthma, inflammatory diseases and chronic obstructive pulmonary disease. The asthma is caused by chronic airway inflammation participated by various cells such as lymphocyte, eosinophil and the like, patients can see uncomfortable symptoms such as wheezing, shortness of breath, chest distress, cough and the like, and partial patients have poor conventional treatment effects, and have the characteristics of long disease course and easiness in repetition, and influence on the life quality of the patients. Inflammatory diseases include tracheitis, bronchitis, pneumonia, and the like, are nonspecific inflammations of the tracheal or bronchial mucosa and surrounding tissues, and are mostly caused by microbial infections such as bacteria, viruses, and the like. Chronic obstructive pulmonary disease is a disease characterized by persistent respiratory symptoms and restricted airflow, caused by toxic particles or gases that cause airway and or alveoli abnormalities. Respiratory symptoms, including dyspnea, cough and/or expectoration are most common, with high incidence and risk of death.
Human umbilical cord blood stem cells are composed mainly of hematopoietic cells, mesenchymal progenitor cells, neural stem/progenitor cells, and endothelial cells. Human umbilical cord blood stem cells can produce large amounts of neurotrophic and angiogenic factors, and the micro-nucleic acids contained in their secreted exosomes. Hematopoietic stem cells have multiple differentiation potential, and endothelial cells can be induced to differentiate from cell subsets by selecting cell subsets from bone marrow and peripheral blood stem cells using cell markers (e.g., CD34, CD14, CD 133).
The stem cell therapy is to transplant healthy stem cells into a patient body so as to achieve the effects of tissue regeneration and repair and organism immunoregulation, and has the characteristics of wide sources, low immunogenicity and strong regeneration and immunoregulation potential. Compared with the traditional therapeutic drugs, the stem cell therapy can inhibit airway inflammation of respiratory diseases by downregulating T cell differentiation, inflammatory factor expression, inhibiting IL-17A secretion, promoting Treg differentiation and other multiple pathways, and improve airway remodeling.
Chinese patent CN202110431148 discloses a pharmaceutical composition containing stem cell vesicles and its use in the treatment of acute and chronic airway inflammation. The stem cell extracellular vesicles described in this patent contain bioactive factors associated with anti-inflammatory or tissue injury repair, including matrix metalloproteinase 1 and hepatocyte growth factor. The patent discloses that the pharmaceutical composition composed of the stem cell extracellular vesicle preparation and the pharmaceutically acceptable carrier can inhibit airway inflammatory response, repair airway injury, improve airway tension and the like when treating airway inflammation.
Chinese patent CN202010365965.6 discloses umbilical cord mesenchymal stem cells for treating pulmonary diseases and a preparation method thereof. The invention provides an umbilical cord mesenchymal stem cell for preparing a medicine for preventing or treating pneumoconiosis and fibrosis, which is applied to treating lung diseases, especially pneumoconiosis and fibrosis, has high safety, good intervention and treatment effects on corresponding diseases and has better application prospect.
Chinese patent CN202210704813.3 discloses the application of dental pulp mesenchymal stem cells in preparing medicament for treating respiratory tract flaccidity. By constructing a rat tracheal flaccidity model, the invention observes that dental pulp mesenchymal stem cells have obvious treatment effect on respiratory tract flaccidity, promotes repair and healing of flaccid oral tissues, reduces lung inflammation, promotes collagen fibrous tissue proliferation and angiogenesis at flaccid oral sites, and provides a stem cell treatment method with convenient material taking and low immunogenicity.
It would be desirable to provide a stem cell therapy that is effective in treating respiratory disorders.
Disclosure of Invention
In order to solve the problems, the invention provides a new application of umbilical cord blood hematopoietic stem cells, in particular to an application for preparing a preparation for treating respiratory diseases.
In one aspect, the invention provides an application of umbilical cord blood hematopoietic stem cells in preparing a preparation for treating respiratory diseases, wherein the preparation method of the umbilical cord blood hematopoietic stem cells comprises the step of adding stem cell separation liquid into umbilical cord blood, wherein the stem cell separation liquid comprises, by weight, 0.1-0.5 part of tea saponin, 2-10 parts of hydroxymethyl cellulose, 4-8 parts of polysucrose and 0.1-1 part of ammonium chloride.
Preferably, the stem cell separating liquid comprises 0.2-0.4 part of tea saponin, 4-6 parts of hydroxymethyl cellulose, 5-7 parts of polysucrose and 0.5-1 part of ammonium chloride in parts by weight.
Further preferably, the stem cell separating liquid comprises 0.3 part of tea saponin, 5 parts of hydroxymethyl cellulose, 6 parts of polysucrose and 0.8 part of ammonium chloride in parts by weight.
Preferably, the respiratory disease includes asthma, tracheitis, bronchitis, pneumonia and chronic obstructive pulmonary disease.
Preferably, the concentration of the cord blood hematopoietic stem cells in the preparation is 10 5 -10 8 cell/mL。
Further preferably, the cord blood hematopoietic stem cells are cord blood mononuclear cells, and the proportion of the hematopoietic stem cells is more than 1%.
Preferably, the preparation of the cord blood hematopoietic stem cells comprises the following steps:
1) Adding stem cell separating liquid into umbilical cord blood;
2) Centrifuging, and sucking a white cloud-like nucleated cell layer between the serum layer and the separation layer.
3) Washing with buffer solution, centrifuging, and incubating the cells to obtain the cord blood hematopoietic stem cells.
Preferably, the collecting of the umbilical cord blood in the step (1) includes the steps of: after the placenta of the lying-in woman is shed, a blood collection bag containing anti-condensation liquid is used, and a needle head on the blood collection bag is inserted into an umbilical vein, so that the anti-condensation liquid is fully mixed with umbilical cord blood, and an umbilical cord blood pretreatment sample is obtained.
Further preferably, the collected cord blood is subjected to ABO/Rh blood group, HLA typing, microbiological detection.
Preferably, the step (1) further comprises a pretreatment step of the umbilical cord blood, specifically, taking the umbilical cord blood, centrifuging for 10 minutes under the condition of 800-1000g, taking the precipitate, and washing out the precipitate with an equal amount of physiological saline or DMEM medium.
Preferably, the volume ratio of the umbilical cord blood to the stem cell separation liquid in the step (1) is 1: (1-2);
further preferably, the volume ratio of the cord blood to the stem cell separation liquid in the step (1) is 1:1.
preferably, the temperature of centrifugation in step (2) is 18-22 ℃;
further preferably, the temperature of the centrifugation in step (2) is 20 ℃.
Preferably, the centrifugation in the step (2) is performed for 15min under the condition of 1500 r/min.
Preferably, the buffer in step (3) includes, but is not limited to, PBS buffer.
Preferably, the temperature of centrifugation in step (3) is 24-26 ℃;
further preferably, the temperature of centrifugation in step (3) is 25 ℃.
Preferably, the rotational speed of the centrifugation in the step (3) is 600-900r/min;
further preferably, the rotational speed of the centrifugation in the step (3) is 800r/min.
Preferably, the centrifugation in the step (3) is carried out for 3-8min;
further preferably, the centrifugation time in the step (3) is 4min.
Preferably, the temperature of the cells incubated in step (3) is 35-40deg.C, in the presence of 5-6% CO 2 Incubating in an incubator for 3-4 hours;
further preferably, the temperature of the cells incubated in step (3) is 37℃in the presence of 5% CO 2 Incubation was performed in an incubator for 3.5h.
Preferably, the step (3) further comprises a step of suspending the cells with physiological saline after the incubation is completed.
In another aspect, the invention provides a pharmaceutical formulation for treating respiratory diseases, the pharmaceutical formulation comprising the cord blood hematopoietic stem cells described above.
Preferably, the concentration of the cord blood hematopoietic stem cells is 10 5 -10 8 cell/mL。
Further preferably, the cord blood hematopoietic stem cells are cord blood mononuclear cells, and the proportion of the hematopoietic stem cells is more than 1%.
Preferably, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.
Further preferably, the pharmaceutically acceptable carrier includes, but is not limited to, emulsifying agents, disintegrating agents, wetting agents, conditioning agents, solubilizing agents, diluents, binders.
Further preferably, the mode of administration of the pharmaceutical formulation includes oral administration and injection administration.
The oral administration pharmaceutical dosage forms include, but are not limited to, powders, tablets, granules, capsules, suspensions.
The dosage forms for injection administration include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavity injection.
The invention has the beneficial effects that:
the umbilical cord blood stem cells provided by the invention are effective in treating respiratory diseases. The new application of the cord blood hematopoietic stem cells provided by the invention has a better application prospect for treating respiratory diseases.
The umbilical cord blood mononuclear cell preparation used in the invention is used for treating respiratory diseases, and does not need blood matching type and HLA matching type.
Drawings
Fig. 1 is a graph of airway resistance parameters Penh for various groups of animals at different methacholine concentrations.
FIG. 2 is a graph of Penh% for each group of animals at different methacholine concentrations.
Fig. 3 is a graph of body weight change for rats in each group, p < 0.05 compared to the normal control group; and (3) compared with a model control group, the #p is less than 0.05.
Fig. 4 is a graph of pulmonary fibrosis scores for rats in each group, p < 0.05 compared to the normal control group; and (3) compared with a model control group, the #p is less than 0.05.
Fig. 5 is a graph of the alveolar inflammatory scores of the rats of each group, p < 0.05 compared to the normal control group; and (3) compared with a model control group, the #p is less than 0.05.
FIG. 6 is a graph of TGF- β1 changes in lung tissue of rats in each group, p < 0.05 compared to normal control group; and (3) compared with a model control group, the #p is less than 0.05.
Fig. 7 is a graph of the HYP change in lung tissue of rats in each group, p < 0.05 compared to the normal control group; and (3) compared with a model control group, the #p is less than 0.05.
FIG. 8 is a graph of MMP-2 change in lung tissue of rats of each group, p < 0.05 compared to the normal control group; compared with the model control group, the #p is less than 0.05; compared with the BM-MSCs transplanted group, the layer p is less than 0.05.
FIG. 9 is a graph showing the variation of TMP-1 in lung tissue of rats in each group, p < 0.05 compared with the normal control group; and (3) compared with a model control group, the #p is less than 0.05.
FIG. 10 is a graph of MMP-2/TMP-1 change in lung tissue of rats in each group, p < 0.05 compared to the normal control group; compared with the model control group, the #p is less than 0.05; compared with the BM-MSCs transplanted group, the layer p is less than 0.05.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of umbilical cord blood hematopoietic Stem cells
The preparation of the cord blood hematopoietic stem cells comprises the following steps:
(1) Fresh cord blood is selected, centrifuged at 800r/min for 8 minutes, whole blood is collected after centrifugation, and the whole blood is placed into a new centrifuge tube, and red blood cells are discarded.
(2) Adding 25ml of stem cell separating liquid into a 50ml centrifuge tube, wherein the stem cell separating liquid comprises 0.3 part of tea saponin, 5 parts of hydroxymethyl cellulose, 6 parts of polysucrose and 0.8 part of ammonium chloride in parts by weight, the volume ratio of the umbilical cord blood to the stem cell separating liquid is 1:1, slowly adding the blood obtained in the step (1) into the upper layer of the separating liquid, and placing the centrifuge tube with the blood and the separating liquid into a centrifuge at the temperature of 25 ℃ at the rotating speed of 1500r/min for 15 minutes.
(3) Taking out the centrifuge tube after centrifugation, wherein the centrifuge tube is divided into 4 layers, namely: a plasma layer, a cell layer, a separation liquid layer, and a red blood cell layer; sucking a white cloud nucleated cell layer between the serum layer and the separation layer, placing the sucked cells into a clean centrifuge tube, and adding PBS to prepare suspension.
(4) The extracted stem cells were washed 3 times with PBS solution and centrifuged at 800r/min for 4 minutes. Washing the centrifuged cells, placing the cells at 37℃and 5% CO 2 Is incubated for 2.5h in the incubator. After centrifugation, cells were suspended in physiological saline and then counted.
Example 2 preparation of umbilical cord blood hematopoietic Stem cells
The preparation of the cord blood hematopoietic stem cells is different from example 1 in that the stem cell separating liquid comprises 0.4 part of tea saponin, 4 parts of hydroxymethyl cellulose, 5 parts of polysucrose and 0.5 part of ammonium chloride according to parts by weight, the volume ratio of the cord blood to the stem cell separating liquid is 1:2, and the rest are the same.
Comparative example 1
The difference from example 1 is that no tea saponin was added to the stem cell isolate, and the remainder was the same.
Comparative example 2
The difference from example 1 is that no ammonium chloride was added to the stem cell isolate, and the remainder was the same.
Comparative example 3
The difference from example 1 is that no hydroxymethyl cellulose was added to the stem cell isolate, and the rest was the same.
1. Detecting the cell activity rate of the prepared cord blood hematopoietic stem cells;
2. the testing method comprises the following steps:
the centrifuged cell suspension was mixed with trypan blue solution at a ratio of 9:1, 10. Mu.l was dropped into a cell counting plate, and living cells and dead cells were counted, and then the cell viability was counted.
Cell viability = total viable cells/(total viable cells + total dead cells) ×100%.
The detection results are shown in Table 1 below.
TABLE 1 cell viability
| Cell viability (%) | |
| Example 1 | 93.6 |
| Example 2 | 93.3 |
| Comparative example 1 | 85.9 |
| Comparative example 2 | 87.1 |
| Comparative example 3 | 86.4 |
As can be seen from Table 1 above, the cell viability of the cord blood hematopoietic stem cells produced by the production method of examples 1-2 was higher than that of comparative examples 1-3, no tea saponin was added to the stem cell isolate of comparative example 1, no ammonium chloride was added to the stem cell isolate of comparative example 2, and no hydroxymethyl cellulose was added to the stem cell isolate of comparative example 3, both affecting the cell viability of hematopoietic stem cells to some extent.
3. Flow cytometer detection: after washing cells with PBS, PE-CD34, PE-CD133, PE-CD90, PE-CD44, PE-CD45 and monoclonal antibodies were added to the PBS-suspended cell suspensions, respectively, and incubated at 4℃for 40min, and the cells were washed and examined by flow cytometry. Meanwhile, negative control antibodies marked by PE-IgG1 and FITC-IgG1 are provided, and stem cells added with monoclonal antibodies are used as blank controls.
Experimental results:
cell count:
the calculation formula is as follows:
note that: in this experiment, the amount of cord blood added per centrifuge tube was 10mL.
Cell count results after calculation, cell count obtained from cord blood was isolated: the number of nucleated cells obtained per mL of cord blood was 3.46×10 5 And each.
Flow cytometry detection shows that the proportion of CD34+ and CD133+ cells in cord blood mononuclear cells (UC-MNCs) is greater than 1.0%.
Experimental example 3 pharmacodynamic experiments of umbilical cord blood mononuclear cells for treating mouse asthma
After the umbilical cord blood mononuclear cells are intravenously injected into the tail of the asthma model BALB/c mouse, the effect of treating asthma is observed, so that the effectiveness of treating the asthma of the mouse by the umbilical cord blood mononuclear cells is determined.
1. Experimental materials and methods
1.1. Material
1.1.1 ovalbumin (OVA, SIGMA), aluminum hydroxide adjuvant for injection (Thermo Fisher Scientific). Phosphate buffer (PBS Gibco) saline (0.9% sodium chloride solution), guangxi Yuan pharmaceutical Co., ltd. Methacholine (MCh, metacholine, SIGMA).
1.1.2. Mice, strain: BALB/c, SPF grade, male. Body weight at test: 20.1-23.0g, the weight difference is within 20% of the average weight. Source and license: beijing Vitolihua laboratory animal technology Co., ltd., laboratory animal production license number is: SCXK 2016-0006, issued by the scientific and technical Committee of Beijing, city. Experimental animal qualification number: 1100112011009108.
1.1.3 environmental conditions of the test: quarantine room and feeding room environments: the room temperature is 20-26 ℃ and the relative humidity is 40-70%. Experimental animal use license number: SYXK (yue) 2016-0122, animal set of standard use: 00233834.
1.1.4 method for modeling the asthma model of mice: the antigen solution for sensitization was administered by intraperitoneal injection at 0.2 mL/dose (the antigen solution was a mixed solution of ovalbumin and aluminum hydroxide adjuvant for injection in equal volumes) on days 1, 7 and 14, and then 10 μl/dose of the antigen solution (ovalbumin solution) was administered by nasal drip at 2 sides of the vehicle after anesthesia with isoflurane on days 21, 22, 23 and 24, and physiological saline was administered in the same manner as the blank.
1.1.5 preparation of human umbilical cord blood mononuclear cells comprising the steps of: experimental example 2 was taken to prepare human umbilical cord blood hematopoietic stem cells. Thawing in water bath at 37deg.C for resuscitation, washing with sodium chloride injection, and re-suspending with sodium chloride injection to obtain suspension containing 10 6 cell/mL cell suspension.
1.1.6 preparation of murine umbilical cord blood mononuclear cells comprising the following steps: experimental example 3 was taken to prepare murine cord blood hematopoietic stem cells. Thawing in water bath at 37deg.C for resuscitation, washing with sodium chloride injection, and re-suspending with sodium chloride injection to obtain the final product 10 6 cell/mL cell suspension.
1.2. Test design
Test dose design and grouping: 140 BALB/c mice which are qualified by quarantine and reach the standard are selected and randomly divided into 7 groups, namely a blank group (1 group), a model group (2 group), a glucocorticoid group (3 group), a low dose group (4 group), a medium dose group (5 group), a high dose group (6 group) and a mouse stem cell group (7 group), wherein 20 groups are respectively (see table 2), and all 4-6 groups are human umbilical cord blood mononuclear cells.
Table 2 test group and dose setting
1.3. Detection method
Noninvasive detection of airway responsiveness (performed in the national emphasis laboratory for respiratory diseases): and (3) taking 1/2 amount of animals from each group within 24h of final excitation to perform noninvasive pulmonary function detection, putting the mice to be detected into a BUXCO noninvasive small animal pulmonary function detection system body drawing box, sequentially atomizing and inhaling 0, 1.56, 3.125, 6.25 and 12.5mg/mL methacholine solution for 30s for stimulation, observing and recording the value of the parameter Penh positively related to the airway resistance for 3min, and continuing atomizing stimulation of the next concentration after resting for 1 min. 4-8 animals were tested in parallel within 40min, euthanized the day after completion, and lung tissue was taken for histopathological examination.
Cell count and classification of alveolar lavage fluid: 1/2 of each group was taken and subjected to tracheal intubation (no collarbone, only the chest was exposed), total lung lavage was performed on mice using 0.7 mL/secondary saline, 3 times in succession, and the recovered alveolar lavage fluid (BALF) was pooled, centrifuged at 2000rpm for 10min, and the lower layer of fluid was taken for 0.3mL, and after shaking, total cell and leukocyte differential counts were performed using an animal-specific hematology analyzer.
Cytokine detection: BALF supernatant was used to detect IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17 cytokine levels using a liquid phase chip (luminex), and TSLP was detected using ELISA (related kit) methods and procedures that are conventional in the art.
Lymphocyte subpopulation detection: the supernatant after BALF centrifugation was taken at 0.3mL and assayed for T cells, th cells (CD4+), tc cells (CD8+), th1, th2, treg, th17 using a flow cytometer.
Histopathological examination: taking animal lung tissues subjected to noninvasive lung function detection, and respectively perfusing left and right lungs (containing bronchi) with 10% neutral formalin solution and Carnoy fixing solution; then, bronchi and lung tissues (two large lung lobes) were taken, pathological tissue sections were prepared, HE staining and PAS staining were performed, respectively, histopathological examination was performed under a microscope, and evaluation was performed using semi-quantification, and the scoring criteria are shown in table 3.
TABLE 3 pulmonary tissue abnormality change scoring criteria
1.4. Data statistical processing method
Statistical analysis was performed on the data of body weight, airway responsiveness non-invasive test results, cell counts of alveolar lavage fluid, cytokines, lymphocyte subpopulations, etc. expressed as Mean ± standard deviation (Mean ± SD), using SPSS statistical software 25.0, first performing a variance alignment test on each set of data using Homogeneity of Variance, if the variance is aligned, performing One-way ANOVA, and if the ANOVA is statistically significant, further performing statistical analysis on the data using Bonferoni method. If the variance is not uniform, welch's correction is used, and when P < 0.05, the variance is considered statistically significant.
2. Experimental results
2.1. Noninvasive detection of airway responsiveness: noninvasive whole-body plethysmography quantitatively reflects the degree of airflow limitation according to the phenomenon that the expiration phase time is prolonged during asthma, and the expiration pause time is prolonged, namely enhanced expiration pause (Penh), and the Penh value is an indirect index for reflecting airway resistance. The values of the parameters Penh positively correlated to airway resistance stimulated by solutions of different concentrations of methacholine (Mch) are shown in table 4, table 5 and fig. 1, and the% Penh is shown in fig. 2.
Table 4 Penh results of animal airway response assays for each group (χ±s, n=10)
Note that: 1. compared to the model group, x represents significant difference p <0.05, x represents significant difference p < 0.01.
2. The glucocorticoid group and the high dose group had 9 animals and the murine stem cell group had 6 animals.
Table 5 results of Penh% for animals tested for airway responses in each group (χ±s, n=10 compared to PBS
Note that: compared to the model group, x represents significant difference p <0.05, x represents significant difference p < 0.01.
The glucocorticoid group and the high dose group had 9 animals and the murine stem cell group had 6 animals.
As can be seen from tables 4-5 above and figures 1-2, the Penh values and Penh% for animals in the blank, glucocorticoid group were significantly lower than those in the model group (P < 0.01) at different methacholine concentration stimuli; the Penh value and/or Penh% of the animals in the low-dose group are significantly lower than those in the model group (P <0.01 or < 0.05) under the stimulation of the concentration of 1.56-6.25mg/mL of methacholine, and the animals have a decreasing trend under the stimulation of the concentration of higher methacholine; the Penh values of animals in the medium dose group and the murine stem cell group are lower than those of animals in the model group under the stimulation of different acetylcholine concentrations, but have no obvious difference; penh% of murine stem cell group animals were significantly lower than model group (P < 0.05) at 1.56mg/mL methacholine concentration; neither Penh values nor Penh% were significantly different from the model group at different methacholine concentration stimulation in the animals of the high dose group (P > 0.05).
2.2. Cytokine detection results: IL-5, IL-6, IL-10, IL-13 were significantly reduced in both the blank and glucocorticoid group BALF (P <0.01 or 0.05), IL-5, IL-13 were significantly reduced in both the medium dose group BALF (P <0.05 or 0.01), and IL-5 was significantly reduced in the low dose group BALF (P < 0.05) compared to the model group; the levels of cytokines for BALF were not significantly different for the remaining animals (P > 0.05). Results for significant reductions in both serum IL-5 and IL-13 (P < 0.01) in the blank and glucocorticoid groups, and significant reductions in IL-6, IL-10 and IL-17 (P <0.01 or 0.05) in the blank group are shown in Table 6 and Table 7. The TSLP of BALF was measured by ELISA for each animal, and was not measured for each group.
Table 6 results of cytokine levels in alveolar lavage fluid from each group of animals (χ.+ -. S, n=5, pg/mL)
Note that: compared to the model group, x represents a difference significance p <0.05, x represents a difference very significant p <0.01.
TABLE 7 serum cytokine concentration (χ.+ -. S, n=5, pg/mL)
Note that: compared to the model group, x represents a difference significance p <0.05, x represents a difference very significant p <0.01.
Table 8 results of lung histopathological examination scoring (Mean ± standard deviation (Mean ± SD), n=10) for each group of animals
Note that: compared to the model group, x represents a difference significance p <0.05, x represents a difference very significant p <0.01.
BALB/c mice were challenged 3 times with ovalbumin, continuously nasal-instilled for 4 days, and non-invasive lung function detection (Penh value), cell and cytokine detection of alveolar lavage fluid, and lung histopathological examination after euthanasia were performed within 24 hours of the last challenge, and comprehensive analysis of the results was concluded as follows.
1. Human umbilical cord blood mononuclear cells (4×10) 5 cell/cell, 2X 10 6 cell/day 7, 20 each) was effective in treating BALB/c mouse asthma.
2. As syngeneic cells, fetal mouse cord blood mononuclear cells (2×10 6 cell/day 7, 20 each) was effective in treating BALB/c mouse asthma.
Experimental example 4 therapeutic Effect of umbilical cord blood Stem cells on rat pulmonary fibrosis
And (3) establishing a pulmonary fibrosis animal model, and observing the influence of umbilical cord mesenchymal stem cells and umbilical cord blood stem cells on rat pulmonary fibrosis.
2. Materials and methods
2.1 test drug:
2.1.1 human cord blood mononuclear cells (CB-MNCs).
2.1.2 rat bone marrow mesenchymal stem cells (BM-MSCs).
2.2 physiological saline, production unit: sichuan Megao Kang Huakang pharmaceutical Co.Ltd
2.3. Test animals and feeding conditions
3.1. Test animals: SPF-class SD rats, 130-200g, male. Provided by the university of Zhongshan laboratory animal center, the laboratory animal production license number is: SCXK (Guangdong) 2011-0029 issued by the scientific and technical parlor of Guangdong province.
3.2. Animal feeding management
3.2.1 animal feeding environment: the temperature is 20-26 ℃; humidity 40% -70%, ventilation times: the feeding chamber was greater than 15 times/hour.
3.2.2 feed: SPF-class sterilized feed for mice.
3.2.3 drinking water: 121 ℃ (1.0 kg/cm) 2 ) Sterilizing tap water for 30min, and feeding water by the following method: can be taken by drinking water bottle.
2.4. Main instrument and reagent
2.4.1. The main instrument is as follows: electric heating constant temperature water tank: DK-8D, haier vertical ultra-low temperature preservation box refrigerator: DW-86L626, taylor-Wharton low pressure liquid nitrogen tank: DPL452-186-0.69I, BHC-1300II A2 biological safety cabinet: BHC1300 II A2, samer femto cell incubator: 3111, sireim high-speed refrigerated centrifuge: ST 16R, inverted study microscope: eclipse Ti2-U, full-automatic biochemical analyzer, pathological section machine.
2.4.2. The main reagent comprises: bleomycin (BLM), cytokine ELISA detection kit: transforming growth factor-beta 1 (TGF-beta 1), hydroxyproline (HYP), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase tissue inhibitor-1 (TIMP-1).
2.5. Test design
2.5.1 moulding: solvent control group 12, model group 39. The groupings are shown in Table 9.
TABLE 9 grouping of animals during molding
2.5.2. Moulding method
2% pentobarbital solution the rats of each group were anesthetized by intraperitoneal injection at 50mg/kg body weight, the trachea was exposed to the incision in the middle of the neck, and the rats of the model group were respectively injected with 0.1mL of BLM physiological saline solution prepared at 5mg/kg body weight in the trachea (solvent control group rats were injected with 0.1mL physiological saline in the trachea), and immediately after the injection, the rats were rotated upright for 2min to uniformly distribute the drug in both lungs. Suturing skin, sterilizing locally, and feeding in original feeding environment.
2.5.3. And (3) detecting after molding: after molding, 3 animals in the model group are randomly taken for blood sampling, dissected and measured for blood biochemical and pathological indexes.
2.5.4 trial grouping and dose setting: after the molding period is finished, the solvent control group animals directly enter the formal test. Model animals were randomly divided into 3 groups of 12 animals each by body weight. The specific experimental groupings and dose designs are shown in table 10.
Table 10 test group and dose design
The administration method comprises the following steps: the tail vein of the medicine is injected,10 6 cells/unit, 24h single injection after modeling.
2.5.5 cell preparation:
2.5.5.1. cord blood mononuclear cells: experimental example 2 was taken to prepare umbilical cord blood hematopoietic stem cells. Thawing in water bath at 37deg.C for resuscitation, washing with sodium chloride injection, and re-suspending with sodium chloride injection to obtain suspension containing 10 6 cell/mL cell suspension.
2.5.5.2. Rat bone marrow mesenchymal stem cells: the rat is sacrificed by neck fracture, the femur of the rear leg is taken under the aseptic condition, the muscle is removed, and the rat is broken from the middle; sucking culture medium with a syringe, blowing into bone cavity to flush bone marrow, centrifuging for 200g and 5min; the culture medium was added and resuspended and inoculated into a 10cm dish. The cells were passaged after confluence by changing the fluid every 2-3 days until about 90% of the cells were grown. Transferring to third generation cell collection, washing, and re-suspending with sodium chloride injection to obtain suspension containing 10 6 cell/mL cell suspension.
2.6. Inspection index
2.6.1. General symptomatic observation: observation time: observations were made 1 day in the morning. The content is observed: death or dying of animals, mental state, behavioral activity, fecal character, feed and drinking water supply, etc.
2.6.2. Weight of: the time was measured and 1 time per week after molding.
Necropsy of 2.6.3 system
Animals were anesthetized by intraperitoneal injection of 20% uliose at 0.7ml100g after the end of the trial. Bleeding and killing, systematic dissection is performed, and pathological changes such as the size, color, texture, section condition and the like of the lung are observed.
2.6.4 sample collection
Acquisition time: half of each group of rats were sacrificed for sampling after modeling 28d and 42d, respectively.
The operation steps are as follows: taking the left lung, washing the left lung by normal saline, and then soaking the left lung in 4% paraformaldehyde buffer solution for fixation for later use; taking 1.000g of right lung and cutting; adding 9mL of precooled PBS solution, and uniformly blowing; homogenizing with a high-speed homogenizer at 15000 rpm; centrifuging the obtained homogenate at 4 ℃ and 2000rpm for 15min; the supernatant was collected at 500uL and stored frozen at-80 ℃.
2.6.5 detection of the distribution of tracer cells in lung tissue: fresh lung tissue was embedded with 10% agar and then subjected to shaking section, and fluorescent cell distribution was observed under a fluorescent inverted microscope.
2.6.6 determination of Lung tissue transforming growth factor- β1 (TGF-. Beta.1), hydroxyproline (HYP), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase tissue inhibitor-1 (TIMP-1) levels: the enzyme-linked immunosorbent assay (ELISA) double-antibody sandwich method is adopted, and simultaneously the TGF-beta 1, HYP, MMP-2 and TIMP-1 levels of the lung tissues of each group of rats are detected in batches, and the TGF-beta 1, HYP, MMP-2 and TIMP-1 levels of the lung tissues of the rats are detected by using the steps and methods conventional in the field.
2.7. Statistical analysis:
for each of the measured data (animal body weight, alveoli score, fibrosis score and organ coefficient) the mean ± standard deviation was calculated and processed using SPSS statistical software. According to different data types, different processing methods are adopted, and when P is less than 0.05, the difference is considered to have statistical significance. The pathological examination is abnormal, and the occurrence rate and the degree of the abnormal condition are compared among groups. The mean comparisons of the groups used either a one-way anova or a Kruskal-Wallis rank sum test, the group-by-group comparisons used a t-test, and the relationship between the two variables used a Spearman rank correlation analysis.
3. Results
3.1. General symptomatic observation:
group a generally had good results with no mortality. B. C, D groups respectively dead 2, 1 and 3 in 7d after modeling and MSCs transplantation, and C, D stem cell transplantation groups of rats have the spirit, activity and diet superior to those of the B groups in the same period, and the difference between the groups is not obvious. All the rat anterior cervical wounds naturally heal after 3-4d molding, and have no red swelling and suppuration.
3.2. Weight of body
After model formation, the body weight of the rats decreased and increased, and the body weight increase of the rats in the cell transplantation group was significantly different from that of the model control (p < 0.05) at 28 days (fig. 3), suggesting that the stem cells can improve the survival quality of the rats with pulmonary fibrosis.
3.3. Rat alveolitis and pulmonary fibrosis scores (table 11, fig. 4-5):
TABLE 11 comparison of scores for degree of pulmonary fibrosis in groups of rats (mean.+ -. Standard deviation (mean.+ -. SD))
Note that: aP <0.05 compared to A1; bP <0.05 compared to B1; cP <0.05 compared to C1; dP <0.05 compared to A2; compared to B2, eP <0.05; fP <0.05 compared to C2.
After animal modeling, the lung fibrosis and alveolitis scores are obviously increased (p < 0.05) on 28 days and 42 days, the lung fibrosis degree on the 28 th day is obviously different from that of a control group (p < 0.05) after the bone marrow mesenchymal stem cell treatment, and the lung fibrosis and alveolitis degree at other time points are improved, but the lung fibrosis and alveolitis degree are not statistically obviously different (p > 0.05) from that of a model control group; after cord blood mononuclear cell treatment, the degree of pulmonary fibrosis on day 28, the pulmonary fibrosis on day 42 and the alveoli scores all differed significantly (p < 0.05) compared to the model control group. The results indicate that both the marrow mesenchymal stem cells and the cord blood mononuclear cells can improve the pulmonary fibrosis and the alveolar inflammatory degree, and the cord blood mononuclear cells have obvious effect and are superior to the marrow mesenchymal stem cells.
3.4. Determination of lung tissue TGF- β1 levels: 28d: a1 is lowest, B1 is highest, C1, E1, D1 and F1 are between A1 and B1, and the values are sequentially reduced; e1, D1, F1 are statistically significant (P < 0.05) compared to B1, respectively. 42d: a2 is lowest, B2 is highest, C2, D2 and E2 are between A2 and B2, and the values are sequentially reduced; the differences between C2, D2 and E2 were statistically significant (P < 0.05) compared to group B2. The mean TGF-. Beta.1 values of lung tissue were elevated in rats of group 42d compared to their same group 28d, but the differences were not statistically significant (P > 0.05) (Table 12, FIG. 6).
3.5. Determination of lung tissue HYP levels (table 12): 28d: a1 is lowest, B1 is highest, C1, D1, E1 and F1 are between A1 and B1, and the values are sequentially reduced; c1, D1, E1, F1 were compared with B1, respectively, the differences being statistically significant (P < 0.05). 42d: a2 is lowest, B2 is highest, C2, D2 and E2 are between A2 and B2, and the values are sequentially reduced; the differences were statistically significant (P < 0.05) in group E2 compared to groups B2 and C2, respectively. The mean value of HYP in lung tissue of rats in each group of 42d is higher than that in the same group of 28d, but the difference is not statistically significant (P > 0.05). (Table 12, FIG. 7)
3.6. Determination of MMP-2 levels in lung tissue: 28d: a1 is lowest, B1 is highest, C1, D1, E1 and F1 are between A1 and B1, the values are sequentially reduced, and the difference between the 4 groups is statistically significant (P < 0.05) compared with the difference between the 4 groups and the B1; d1, E1, F1 are statistically different from C1 (P < 0.05), respectively; f1 was statistically significant (P < 0.05) compared to D1, E1, respectively. 42d: a2 is lowest, B2 is highest, C2, E2 and D2 are between A2 and B2, the values are sequentially reduced, and the difference between the 3 groups is statistically significant (P < 0.05) compared with the difference between the 3 groups and the B2; the differences between D2 and E2 compared to C2 were statistically significant (P < 0.05). The average value of MMP-2 in the lung tissue of the rats in the groups 42, D A and C, E is increased compared with that in the same group 28D, and the average value of MMP-2 in the lung tissue of the rats in the groups 42, D B and D is decreased compared with that in the same group 28D (table 12, figure 8).
TABLE 12 TGF-beta 1, HYP, MMP-2, TIMP-1, MMP-2/TIMP-1 comparisons (mean.+ -. Standard deviation (mean.+ -. SD))
Note that: aP <0.05 compared to A1; bP <0.05 compared to B1; cP <0.05 compared to C1; dP <0.05 compared to A2; compared to B2, eP <0.05; fP <0.05 compared to C2.
3.7. Determination of lung tissue TIMP-1 levels:
28d: a1 is highest, B1 is lowest, F1, C1, D1 and E1 are arranged between A1 and B1, and the numerical values are sequentially increased; d1, E1 are statistically different from B1 (P < 0.05), respectively; e1 is statistically significant (P < 0.05) compared to C1, F1, respectively. 42d: a2 is highest, B2 is lowest, D2, C2 and E2 are between A2 and B2, and the values are sequentially increased; e2 is statistically significant (P < 0.05) compared to C2, D2, respectively. The mean TIMP-1 value of the lung tissue of the rats in each group of 42d is reduced compared with that of the rats in the same group of 28 d. (Table 12; FIG. 9)
3.8. Pulmonary tissue MMP-2/TIMP-1:28d: a1 is lowest, B1 is highest, C1, D1, F1 and E1 are between A1 and B1, the values are sequentially reduced, and the difference between the 4 groups is statistically significant (P < 0.05) compared with the difference between the 4 groups and the B1; differences in D1, F1, E1 compared to C1, respectively, were statistically significant (P < 0.05). 42d: a2 is lowest, B2 is highest, C2, D2 and E2 are between A2 and B2, the values are sequentially reduced, and the difference between the 3 groups is statistically significant (P < 0.05) compared with the difference between the 3 groups and the B2; the differences between D2 and E2 compared to C2 were statistically significant (P < 0.05). The mean value of MMP-2/TIMP-1 was increased in the lung tissue of rats in group 42d compared with that in group 28d, but the difference was not statistically significant (P > 0.05) (Table 12; FIG. 10) and there was a significant positive correlation between TGF- β1, HYP, MMP-2/TIMP-1 in lung tissue with the alveolar inflammatory degree score and the pulmonary fibrosis degree score, respectively (Table 13).
TABLE 13 analysis of the correlation between TGF-beta 1, HYP, MMP-2/TIMP-1 and the degree of alveolitis and degree of pulmonary fibrosis, respectively
3.9. Fluorescent cells were observed by shaking section: fluorescent cells were not found after the 28-day and 42-day sacrificed rat lung tissue shake sections, suggesting that differentiation replacement effect of stem cells may not be the main mechanism of action for treating pulmonary fibrosis.
The results indicate that MSCs can delay the progress of pulmonary fibrosis through vein transplantation, the down regulation of TGF-beta 1 level and the improvement of MMP/TIMP imbalance can promote the repair effect of MSCs on the pulmonary fibrosis, and the selection of umbilical cord blood stem cells can obviously improve the treatment effect on the pulmonary fibrosis of rats.
Experimental example 5 therapeutic Effect of umbilical cord blood Stem cells on influenza and severe pneumonia in elderly people
Influenza is an acute febrile respiratory infectious disease caused by influenza virus, and is transmitted by spray, and clinically typical symptoms of systemic poisoning such as protrusion aversion to cold, high fever, headache, systemic soreness, fatigue and the like are shown. Epidemiology is most notable for sudden outbreaks, rapid spread, and broad spread. Influenza is often aggravated 2-4 days after onset or in the recovery period, and has high fever, severe cough, purulent sputum, dyspnea, and lung dampness-type rales. Infant, elderly, patient with cardiopulmonary disease and other chronic diseases or immunocompromised person can complicated pneumonia, prognosis is poor.
1. Materials and methods
1.1 general data
140 hospitalized patients meeting the clinical influenza severe diagnosis standard are collected; 74 cases for men and 66 cases for women; age 60-83 years, mean 69 years. 121 patients with influenza high risk factors account for 86.43%, 62 patients with chronic diseases such as respiratory system and cardiovascular system; 12 obese people. The cells were divided into 70 stem cell groups (treatment group) and 70 comprehensive treatment groups (control group) by the random number table method.
35 men and 35 women in the treatment group; age 66-83 years, median 71 years; 39 men and 31 women in the control group; age 60-82 years, median age 70 years, sex and age group balance.
1.2 diagnostic criteria
1.2.1 diagnosis of influenza severe symptoms:
meets the diagnosis of influenza in the influenza diagnosis and treatment guidelines (2018 edition) and has one of the following serious standards:
(1) Fast breathing rate, dyspnea and cyanosis of the lips;
(2) Mental changes such as slow response, somnolence, agitation, convulsion, etc.;
(3) Severe vomiting, diarrhea, dehydration manifestation;
(4) Imaging with symptoms of pneumonia;
(5) Myocardial enzyme levels such as Creatine Kinase (CK) and creatine kinase isozymes (CK-MB) are rapidly increased;
(6) The original basic disease is obviously aggravated.
1.2.2 diagnosis of bacterial pneumonia secondary to influenza
Patients with severe influenza persist for >3 days with severe cough, purulent sputum, dyspnea, lung wetting-pattern of lung-heat; any of the following detection indexes exceeds the upper limit of the normal value, and the occurrence of secondary bacterial pneumonia can be considered: (1) total peripheral blood leukocytes; (2) peripheral blood neutrophils; (3) C reaction protein.
1.3 inclusion criteria
Meets the diagnosis standard of influenza bacterial pneumonia, and the age is more than or equal to 60 years; the stem cell clinical study was approved and informed consent was signed.
1.4 exclusion criteria
Patients with mental diseases. Other influenza drug clinical researchers have been enrolled for nearly 1 month. Meets one of the critical diagnosis standards: respiratory failure; infection toxic shock; multiple organ dysfunction; other serious clinical situations in which care is required. Pregnant women.
1.6 treatment regimen
1.6.1 control group
Comprehensive treatment is performed by referring to a diagnosis and treatment scheme of influenza A (2010 edition) and a diagnosis and treatment guide of influenza (2018 edition), wherein the comprehensive treatment comprises antiviral (for example, oseltamivir) and symptomatic support, and different antibiotics (for example, penicillin, azithromycin, levofloxacin, amoxicillin, cefaclor) and the like are selected according to the condition of a patient.
1.6.2 treatment group: on the basis of comprehensive treatment, cord blood stem cells (HLA matching is not carried out before the cord blood stem cells are applied, and the ratio is 2 multiplied by 10) 6 Per kg body weight, intravenous drip in 100ml physiological saline).
1.7 observations index
Detecting lung X-ray, blood routine and C-reactive protein to judge the secondary bacterial pneumonia; the use of anti-influenza virus drugs was recorded.
1.8 statistical methods
And (3) establishing a central database input system, respectively inputting data by two persons, and checking the accuracy of the data. Data analysis is carried out by using SAS9.1.3 statistical software, the counting data are described in terms of frequency and percentage, and the difference is statistically significant by adopting rank sum test and P being less than or equal to 0.05.
2. Results
2.1 comparison of the use of two groups of anti-influenza Virus drugs
Of the 70 patients in the treatment group, 46 used antiviral drugs, accounting for 65.71%; 54 of the 70 control groups were used and accounted for 77.14%. The comparison difference of group 2 has no statistical significance. Indicating that the antiviral treatment levels of the 2 groups of patients are consistent.
2.2 comparison of two groups of secondary bacterial pneumonia cases
Among 140 severe influenza patients, 45 cases of secondary bacterial pneumonia occurred, accounting for 32.14%. In the 70 patients in the treatment group, 9 cases of bacterial pneumonia are generated, accounting for 12.86%, and in the 70 cases of the control group, 36 cases of bacterial pneumonia are generated, and the ratio is 51.42%. The incidence of bacterial pneumonia was compared in group 2, and the differences were statistically significant (p < 0.01). It is shown that the umbilical cord blood stem cell treatment can promote recovery of severe influenza and reduce incidence of complicated pneumonia.
Experimental example 6 therapeutic Effect of umbilical cord blood Stem cells on refractory bronchial asthma
Persistent infiltration of inflammatory cells, hyperplasia and hypertrophy of airway smooth muscle cells, reduction of airway caliber, even partial stenosis or blockage, irreversible airflow limitation and finally lung function change occur in the development process of refractory asthma. The traditional medicines for treating refractory bronchial asthma are more, and mainly comprise symptom control medicines and symptom relief medicines.
Severe asthma refers to an acute or explosive episode of asthma that cannot be ameliorated by conventional treatment, is persistent, or enters a critical state for a short period of time and develops into respiratory failure, severely endangering patient life. The severe asthma has complex pathogenesis, poor drug treatment response, high treatment difficulty and high mortality rate. In recent years, animal experiments for stem cell therapy of asthma have been greatly progressed, but are more limited to mesenchymal stem cells. The invention utilizes umbilical cord blood stem cells to treat asthma, obtains better clinical effects, and has the following results:
1 materials and methods
1.1 case data: 126 cases of severe asthma patients were selected and randomized into two groups of 63 cases each. 31 men and 32 women in the stem cell group; age 19-65 years, average age (41.17 ±5.39) years; the disease course is 4-16 years, and the average (6.18+/-2.74) years. 33 men and 30 women in the control group; age 20-64 years, average age (40.16±5.88); the disease course is 3-14 years, and the average (5.63+ -3.17) years. The difference between the two groups is not statistically significant (P > 0.05) and is comparable.
1.2 dosing regimen
The lead-in period is 4 weeks, during which the patient continues to use high dose inhaled glucocorticoid (ICS) conventional therapy (ICS dose must be ≡500 ug/day daily, fluticasone propionate or equivalent (Shu Lidie/250 ug 2 times/day and higher doses or equivalent would meet the ICS standard for ICS/LABA complex).
Stem cell group: after the introduction is finished, when the percutaneous blood oxygen saturation of the patient is more than 92% and the vital sign is stable, the patient is intravenous injected with transplanted cells, HLA matching is not carried out before the application of umbilical cord blood stem cells, and the single-time implantation of human umbilical cord blood mononuclear cells (hUC-MNCs) dose is 2.0-3.0x10 8 (4.5X10 by weight of 60 kg adult) 6 /kg). 2 weeks later, performing a second infusion, and intravenous injection of transplanted cells into the patient when the percutaneous blood oxygen saturation of the patient is above 92% and vital signs are stable, wherein the single implantation dose of hUC-MNCs is 2.0-3.0X10 8 (4.5X10 by weight of 60 kg adult) 6 /kg)。
Control group: after the introduction, 0.9% sodium chloride injection is injected into the patient intravenously when the percutaneous blood oxygen saturation of the patient is above 92% and the vital sign is stable. A second infusion was performed after 2 weeks, and the patient was given an intravenous injection of 0.9% sodium chloride when the patient had a percutaneous blood oxygen saturation of 92% or more and the vital signs were stationary.
( And (3) injection: each group of subjects was given a 100ml washpipe of physiological saline after each infusion, so that the whole of the preparation remaining in the infusion pipe was infused into the patient. )
1.3 efficacy evaluation index: the following indicators were observed at 1 week, 4 weeks and 12 weeks after treatment, respectively.
1.3.1 clinical symptoms (chest pain, shortness of breath, wheezing and chest distress) are basically or completely disappeared, no asthma attack occurs within 1 week, and the sleeping quality is good at night and is obvious; the clinical symptoms are improved, the times of asthma attacks are reduced before, the times are less than or equal to 2 times per week, and the night sleep quality is improved to be effective; clinical symptoms are not improved at all compared with the prior treatment, and the symptoms even have aggravation trend and are ineffective. Total effective rate = significant rate + effective rate.
1.3.2 quality of life: scoring was performed using the asthma quality of life questionnaire (AQLQ standard version).
1.3.3 detection of the partial pressure of arterial blood carbon dioxide (PaCO 2) and partial pressure of arterial oxygen (PaCO) before and after treatment of a patient by blood gas analysis 2 ). The two groups of patients were examined for first second expiratory volume (FEV 1), first second forced expiratory volume as a percentage of forced vital capacity (FEV 1/FVC%) and maximum expiratory flow (PEF) changes before and after treatment using a lung function detector.
1.4 statistical methods data analysis was performed using SPSS20.0 statistical software, metering data were expressed as mean.+ -. Standard deviation (mean.+ -. SD), count data were expressed as% and comparisons of group rates were tested using t or χ2, with a P <0.05 difference statistically significant.
2. Results
2.1. Clinical efficacy comparisons for two groups of patients 12 weeks after treatment: the total effective rate of treatment was 95.23% for the stem cell group, which was compared to the control group (P < 0.01), as shown in table 14, when counted with 74.60% for the control group.
Table 14. Comparative efficacy of two groups of patients (examples (%))
2.2. The results of quality of life comparisons for the two groups of patients at 12 weeks post-treatment are shown in Table 15.
Table 15 comparison of quality of life (AQLQ) scores of asthma in two groups of patients (Mean ± standard deviation (Mean ± SD), n=63
The difference was not obvious in the two groups of pre-treatment comparisons: comparison after treatment of the two groups, p <0.05 compared to the control group.
2.3 blood gas analysis index comparison results of two groups of patients at 12 weeks after treatment are shown in Table 16.
Table 16. Blood gas analysis index comparison (mean.+ -. Standard deviation (mean.+ -. SD), n=63) for two groups of patients
The difference was not obvious in the two groups of pre-treatment comparisons: comparison after treatment of the two groups, p <0.05 compared to the control group.
2.4. The lung function index 12 weeks after both treatment groups are shown in Table 17.
Table 17 comparison of lung function indicators before and after treatment (mean.+ -. Standard deviation (mean.+ -. SD), n=63) for two groups of patients
The difference was not obvious in the two groups of pre-treatment comparisons: comparison after treatment of the two groups, p <0.05 compared to the control group.
Severe asthma is classified into acute severe asthma and acute asphyxia asthma, and gas exchange and hemodynamics are abnormal during attack, so that airway resistance is obviously increased. Hypoxia is the leading cause of severe asthma death, so continuous oxygen inhalation is a direct route to improve hypoxia. Broad-spectrum antibiotics are commonly used for treatment; theophylline drugs can inhibit phosphodiesterase, increase cAMP concentration in smooth muscle cells, and antagonize adenosine receptors; the glucocorticoid can inhibit inflammatory process and inflammatory mediator release, reduce airway reactivity, inhibit chemotaxis and activation of eosinophils, and has antiallergic effect, and can relieve mucosal edema and obstruction. The invention suggests that the cord blood stem cells have obvious effect of treating severe asthma by improving clinical symptoms of severe asthma patients, reducing the times of attacks of asthma, improving ventilation and respiratory symptoms and obviously improving life quality.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The application of the umbilical cord blood hematopoietic stem cells in preparing the preparation for treating the respiratory diseases is characterized in that the preparation method of the umbilical cord blood hematopoietic stem cells comprises the step of adding stem cell separation liquid into umbilical cord blood, wherein the stem cell separation liquid comprises, by weight, 0.1-0.5 part of tea saponin, 2-10 parts of hydroxymethyl cellulose, 4-8 parts of polysucrose and 0.1-1 part of ammonium chloride.
2. The use according to claim 1, wherein the stem cell separating liquid comprises, by weight, 0.2-0.4 parts of tea saponin, 4-6 parts of hydroxymethyl cellulose, 5-7 parts of polysucrose and 0.5-1 part of ammonium chloride.
3. The use according to claim 1, wherein the stem cell separation liquid comprises 0.3 parts by weight of tea saponin, 5 parts by weight of hydroxymethyl cellulose, 6 parts by weight of polysucrose and 0.8 parts by weight of ammonium chloride.
4. The use according to claim 1 wherein the respiratory diseases include asthma, tracheitis, bronchitis, pneumonia and chronic obstructive pulmonary disease.
5. The use according to claim 1, wherein the concentration of cord blood hematopoietic stem cells in the formulation is 10 5 -10 8 cell/mL。
6. The use according to claim 1, wherein said cord blood hematopoietic stem cells are cord blood mononuclear cells having a hematopoietic stem cell fraction of greater than 1%.
7. The use according to any one of claims 1 to 6, wherein the preparation of cord blood hematopoietic stem cells comprises the steps of:
1) Adding stem cell separating liquid into umbilical cord blood;
2) Centrifuging, and sucking a white cloud-like nucleated cell layer between the serum layer and the separation layer;
3) Washing with buffer solution, centrifuging, and incubating the cells to obtain the cord blood hematopoietic stem cells.
8. A pharmaceutical formulation for treating respiratory diseases, comprising umbilical cord blood hematopoietic stem cells of any one of claims 1-7.
9. The pharmaceutical preparation of claim 8, wherein the concentration of cord blood hematopoietic stem cells is 10 5 -10 8 cell/mL。
10. The pharmaceutical formulation of any one of claims 8-9, wherein the cord blood hematopoietic stem cells are cord blood mononuclear cells having a hematopoietic stem cell fraction of greater than 1%.
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