CN116024166A - Human umbilical cord mesenchymal stem cell preparation and preparation method and application thereof - Google Patents
Human umbilical cord mesenchymal stem cell preparation and preparation method and application thereof Download PDFInfo
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- CN116024166A CN116024166A CN202211743191.1A CN202211743191A CN116024166A CN 116024166 A CN116024166 A CN 116024166A CN 202211743191 A CN202211743191 A CN 202211743191A CN 116024166 A CN116024166 A CN 116024166A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of autoimmune disease prevention and treatment, and discloses a human umbilical mesenchymal stem cell preparation and a preparation method and application thereof. The preparation method of the invention comprises the following steps: obtaining primary human umbilical cord mesenchymal stem cells; the primary human umbilical cord mesenchymal stem cells are from neonatal umbilical cord tissue produced by term caesarean section; the age of the mother of the newborn is 20-25; the umbilical cord tissue is an umbilical cord tissue with the length of 5-10 cm cut at the position of 8-12 cm at one end of the umbilical cord neonate; culturing and amplifying the primary human umbilical cord mesenchymal stem cells to obtain passaged human umbilical cord mesenchymal stem cells; and preparing the cell suspension from the passaged human umbilical cord mesenchymal stem cells. The human umbilical mesenchymal stem cells prepared by the invention have excellent curative effect on early or light SLE, have the effects of regulating the disordered immune function and slowing down or changing the disease process, and provide a new treatment strategy for the treatment of early or light SLE.
Description
Technical Field
The invention relates to the technical field of autoimmune disease prevention and treatment, in particular to a human umbilical cord mesenchymal stem cell preparation and a preparation method and application thereof.
Background
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by abnormal activation of B lymphocytes and production of autoantibodies, which bind to corresponding autoantigens and then deposit on skin, joints, glomeruli and other sites, resulting in multiple organ and system damage (e.g., lupus nephritis, LN). Hormone-cyclophosphamide is a classical therapy for SLE treatment, can effectively relieve SLE progression, and improve SLE survival and prognosis, however, has remarkable side effects after long-term administration, and can induce death of patients due to side effects such as infection, myelosuppression, secondary malignant tumor, disease recurrence after drug withdrawal, and the like.
Diagnostic typing of SLE is of great importance for the choice of treatment regimen. B cell targeted biologicals that specifically inhibit B lymphocyte activating factor (BAFF), such as Belimumab (Belimumab), have been shown to alleviate clinical symptoms in SLE patients, reduce the generation of immune abnormalities, and are used in the treatment of moderately severe SLE, but Belimumab, using a single target, cannot inhibit the transformation of plasma cells and memory B cells. At present, how to successfully treat early stage mild SLE patients without side effects remains a great challenge. Treatment of early/light lupus (early being indicative of MRL/lpr lupus mice 8-11 weeks old in animal models (Brain Behav Immun.2002,16 (1): 46-61), and light being indicative of SLE patients (J Rheumatoid. 2002,29 (2): 288-291) clinically diagnosed with a 5.ltoreq.SLEDAI-2000 score of less than 9, includes the use of low doses of corticosteroids and antimalarial drugs (antimalarial drugs), vitamin D, statin drugs, etc. while these drugs appear to be effective in the treatment of early/light SLE, some SLE performance is not fully controlled, i.e., does not sufficiently prevent progression of disease even to End Stage Renal Disease (ESRD). Furthermore, side effects of drugs such as Hydroxychloroquine (HCQ) have been of great concern, and HCQ is also ineffective in preventing the occurrence of severe manifestations.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a human umbilical cord mesenchymal stem cell preparation, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a preparation method of a human umbilical cord mesenchymal stem cell preparation, comprising the following steps:
(1) Obtaining primary human umbilical cord mesenchymal stem cells;
the primary human umbilical cord mesenchymal stem cells are from neonatal umbilical cord tissue produced by term caesarean section; the age of the mother of the newborn is 20-25; the umbilical cord tissue is an umbilical cord tissue with the length of 5-10cm cut at the position of 8-12 cm at one end of the umbilical cord neonate;
(2) Culturing and amplifying the primary human umbilical cord mesenchymal stem cells to obtain passaged human umbilical cord mesenchymal stem cells;
(3) And preparing the cell suspension from the passaged human umbilical cord mesenchymal stem cells.
The umbilical cord of the invention is derived from neonates (length is 40-60 cm) of healthy mothers who are between 20 and 25 years old and have no infectious diseases or genetic diseases and hUC-MSCs prepared from a section of umbilical cord tissue with length of 5-10cm, wherein the position of the umbilical cord is positioned at about 10cm at one end of the neonate (toward the maternal direction), and the hUC-MSCs have higher activity and excellent curative effect.
As a preferred embodiment of the preparation method, in the step (1), the method for obtaining primary human umbilical cord mesenchymal stem cells comprises the following steps:
taking the umbilical cord tissue, cleaning and sterilizing,removing arteries and veins, and cutting to a surface area of about 10-25 mm 2 Standing for 15-30 min, adding culture solution, standing at 37deg.C with 5% CO 2 Culturing under the condition of the culture medium to obtain primary human umbilical cord mesenchymal stem cells.
As a preferred embodiment of the preparation method, in the step (2), the method for culturing and amplifying the primary human umbilical cord mesenchymal stem cells comprises the steps of:
taking the primary human umbilical cord mesenchymal stem cells, adding pancreatin, and digesting at 37 ℃ to obtain a cell suspension; centrifuging the obtained cell suspension, removing supernatant, adding fresh culture solution, resuspending cells, standing at 37deg.C and 5% CO 2 And (5) culturing.
As a preferred embodiment of the preparation method, in the step (3), the method for preparing a cell suspension is as follows:
taking the passaged human umbilical cord mesenchymal stem cells, cleaning, adding pancreatin, digesting at 37 ℃, and filtering by a cell sieve to obtain a cell suspension; centrifuging the obtained cell suspension, removing supernatant, adding cell preparation suspension to resuspend cells, and packaging; the cell preparation suspension is physiological saline containing 1% of human albumin.
In a preferred embodiment of the above production method, in the steps (1) to (3), the medium for the cells is MEM-alpha medium containing 5% serum replacement, and the pH is 7.2 to 7.4.
As a preferred embodiment of the preparation method, PBS containing 0.25% trypsin and no EDTA is used for the digestion.
In a second aspect, the invention provides a human umbilical mesenchymal stem cell preparation prepared by the preparation method.
The human umbilical cord mesenchymal stem cell preparation provided by the invention is weak in immunogenicity and does not induce immune rejection; no further disputes concerning society, ethics, law; the source is sufficient; the separation method is simple, the cell activity is strong, and the amplification is easy; umbilical cord mesenchymal stem cells are derived from human umbilical cord tissue and are a more primitive population of stem cells.
In a third aspect, the preparation method and the human umbilical cord mesenchymal stem cell preparation are applied to the preparation of a transplantation medicine for treating or preventing early or mild systemic lupus erythematosus.
The human umbilical cord mesenchymal stem cell preparation of the invention is used for independently transplanting hUC-MSCs to MRL/lpr mice in early disease stage (8-11 weeks), can obviously inhibit the autoimmune reaction intensity, reduce the serum inflammatory factor level, improve kidney pathology, protect kidney function, and has no side effects such as infection.
As a preferred embodiment of the use according to the invention, the transplantation dosage of the transplantation drug is 0.4X10 6 ～1.2×10 6 Human umbilical cord mesenchymal stem cells/10 g; the infusion times are 1-3 times, and 200-300 mu L each time.
In a fourth aspect, the preparation method of the invention and the human umbilical cord mesenchymal stem cell preparation are applied to the preparation of a medicine for co-culturing human umbilical cord mesenchymal stem cells and peripheral blood B cells of patients with early or mild systemic lupus erythematosus.
Co-culturing hUC-MSCs and peripheral B lymphocytes of patients with mild SLE (SLEDAI-2000 score is less than or equal to 5 and less than or equal to 9) shows that the hUC-MSCs can obviously inhibit peripheral CD138 of patients with mild SLE + CD19 + B cells proliferate, significantly reducing the level of IgG total antibodies produced by B cells. Has the functions of regulating the disordered immunity and slowing down or changing the disease process, has obvious effect on patients, and is safe and effective without any adverse side effect.
Compared with the prior art, the invention has the beneficial effects that:
in the preparation method of the human umbilical mesenchymal stem cells (hUC-MSCs), the prepared hUC-MSCs are derived from neonatal umbilical cords (length is 40-50 cm) of healthy mothers with no infectious diseases or genetic diseases and term co-production, and the position of the umbilical cords is positioned at a position (direction to a mother) of about 10cm at one end of the neonate, and a section of umbilical cord tissue with a length of 5-10 cm. The prepared hUC-MSCs have higher activity, and the MRL/lpr mice after the transplantation of the prepared hUC-MSCs have lower death rate. The invention can be used for treating light SLE by singly transplanting hUC-MSCs, can obviously inhibit the autoimmune reaction intensity, reduce the serum inflammatory factor level, improve the kidney pathology, protect the kidney function and avoid side effects such as infection and the like by singly transplanting hUC-MSCs to light lupus mice (MRL/lpr). The hUC-MSCs of the invention can also inhibit the differentiation of peripheral B lymphocytes of SLE patients and reduce the capacity of B cells to produce antibodies.
In a word, the hUC-MSCs prepared by the invention have excellent curative effect on light SLE, have the effects of regulating the disordered immune function and slowing down or changing the disease process, and are safe and effective without any adverse side effect. Provides a new effective and safe treatment strategy for the treatment of early/mild SLE.
Drawings
FIG. 1 is a statistical plot of the effect of different maternal age and location of the excised umbilical cord on the activity of isolated hUC-MSCs cells, and their effect on mortality in mice after transplantation.
FIG. 2 is a graph showing the results of serum autoantibodies from hUC-MSCs transplantation time axis and 6-week-old MRL/lpr mice.
FIG. 3 is the effect of hUC-MSCs transplantation on MRL/lpr mouse autoantibody levels and spleen weight ratio.
FIG. 4 is a graph showing the effect of hUC-MSCs transplantation on the MRL/lpr mice peripheral blood B cell subtype ratio.
FIG. 5 is a graph showing the effect of hUC-MSCs transplantation on serum inflammatory cytokines in MRL/lpr mice.
FIG. 6 is a graph showing the effect of hUC-MSCs transplantation on kidney function in MRL/lpr mice.
FIG. 7 is a graph showing the effect of hUC-MSCs transplantation on kidney injury in MRL/lpr mice.
FIG. 8 is a graph showing the results of the co-culture of hUC-MSCs with peripheral blood B cells from clinical SLE patients.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, are commercially availableThe industrial route is obtained. The medium for stem cells in the examples was MEM-alpha medium (5% serum replacement, 5% blood substitute was KnockOut) TM Serum replacement, available from ThermoFisher company under Gibco), pH 7.2-7.4. All data in the examples were statistically analyzed using Graphpad Prism 8.0.2 statistical software. The Kolmogorov-Smirnov test and Shapiro-Wilk test were used to determine whether the data met the normal distribution. Unpaired comparison of the two sets of normal distribution data, the general unpaired t test was used when the variance was uniform, and the Welch t test was used when the variance was non-uniform. The normal distribution data between the two groups is compared by adopting paired t test. Average comparison of two or more sets of normal distribution data used One-way ANOVA (One-way ANOVA). The average of two or more sets of normal distribution data was compared using the Brown-forsyth and Welch ANOVA test. Non-paired comparisons between two variables were measured using the Mann-Whitney test, paired comparisons were measured using the Wilcoxon signed rank test, and multivariate comparisons were measured using the Kruskal-Wallis test. Each statistical method P <The differences were considered statistically significant at 0.05.
Example 1: isolation and culture of human umbilical cord mesenchymal stem cells (hUC-MSCs)
(1) Separation
Approved by the ethical committee of the hospital, informed consent of the patient and family members is obtained, and the umbilical cord (length 40-50 cm) of the neonate who is healthy and free of genetic diseases and has term caesarean section of healthy mother is obtained from the affiliated hospital of the medical university of Guangdong. The umbilical cord was placed in a collection bottle containing phosphate buffer (PBS containing 1% diabody, i.e., 100. Mu.g/ml penicillin and 100. Mu.g/ml streptomycin) to be treated. Before operation, the experimental environment is checked, the equipment and reagent information is confirmed, and the required consumables and instruments are transferred into the sterile field of the cell preparation shop as specified. The super clean bench is opened for ventilation, the indicator lamp turns green, and the whole super clean bench is uniformly wiped by sterile gauze containing 75% alcohol, and required appliances, consumables and the like can be put into the super clean bench after being sterilized by 75% alcohol. The dishes, tweezers and scissors were prepared. Sterilizing the outer wall of the collection bottle with 75% alcohol, transferring umbilical cord tissue into a culture dish with sterile forceps, rinsing with pre-cooled PBS buffer solution containing 1% double antibody, and removingRemove bloodstains. A section of umbilical cord tissue with a length of 5-10cm is cut off at a position (toward the mother) of about 10cm at one end of the neonate by sterile cutting, the tissue is cut into sections of 2-3 cm, the blood clot is washed with PBS (1% diabody) buffer solution, and the procedure is repeated for 2-3 times until the washing solution is clear. And cutting off the umbilical cord, and sequentially removing arteries and veins. Cutting tissue to a surface area of about 10-25 mm 2 Is spread on the bottom surface of a culture dish, is kept stand for 15 to 30 minutes, is slowly added with a proper amount of culture medium along the side wall, is placed at 37 ℃ and is subjected to 5 percent CO 2 Culturing in an incubator. The fresh culture medium was changed 1 time every 3 days, and the growth of the cells was observed under a microscope, and when the cells were grown to 80% confluence, the cells were digested with 0.25% pancreatin (no EDTA was necessary) and passaged for 1 to 2 minutes, and the cells were designated as first-generation cells (P1).
(2) Culturing
The super clean bench is opened for ventilation, the indicator lamp turns green, and the whole super clean bench is uniformly wiped by sterile gauze containing 75% alcohol, and required appliances, consumables and the like can be put into the super clean bench after being sterilized by 75% alcohol. Taking out primary cells needing to be changed from the incubator, and putting the primary cells into an ultra-clean bench. The culture solution in the culture dish is sucked by a pipette and discarded in a waste liquid jar. To each dish was added the prepared culture solution. After all the culture dishes are filled with liquid, the culture dishes are marked with the date of liquid change, and the culture dishes are placed into an incubator.
(3) Passage of
Before operation, the experimental environment is checked, the equipment and reagent information is confirmed, and the required consumables and instruments are transferred into the sterile field of the cell preparation shop as specified. The super clean bench is opened for ventilation, the indicator lamp turns green, and the whole super clean bench is uniformly wiped by sterile gauze containing 75% alcohol, and required appliances, consumables and the like can be put into the super clean bench after being sterilized by 75% alcohol. And taking out cells to be passaged from the incubator, putting the cells into an ultra-clean bench, sucking culture solution in a culture dish by using a pipettor, and discarding the culture solution in a waste liquid tank. Each dish was washed with 0.9% physiological saline. Pancreatin was added to each dish and digested at 37 ℃. After the digestion of the cells in all dishes is completed, stop solution is added to terminate the digestion. The cell suspension was transferred to a 50mL centrifuge tube and the supernatant was discarded by centrifugation. Cells were resuspended in an appropriate amount of culture medium and counted. The cell suspension was seeded into a new dish according to the counting result. The culture dish was marked with the number of the collected cells, the passage date, and the number of the cell passages, and then placed in an incubator.
(4) Freezing and preserving
Before operation, the experimental environment is checked, the equipment and reagent information is confirmed, and the required consumables and instruments are transferred into the sterile field of the cell preparation shop as specified. The super clean bench is opened for ventilation, the indicator lamp turns green, and the whole super clean bench is uniformly wiped by sterile gauze containing 75% alcohol, and required appliances, consumables and the like can be put into the super clean bench after being sterilized by 75% alcohol. Taking out the cells to be frozen from the incubator, placing the cells in the middle of the super clean bench, sucking the culture solution in the culture dish by using a pipettor, and discarding the culture solution in a waste liquid tank. Each dish was washed with 0.9% physiological saline. Pancreatin was added to each dish and digested at 37 ℃. After the digestion of the cells in all dishes is completed, stop solution is added to terminate the digestion. The cell suspension was transferred to a 50mL centrifuge tube, counted, and the supernatant was discarded by centrifugation. Preparing cell freezing solution. And (3) adding the prepared frozen stock solution to resuspend the cells, and filling the cell suspension into a frozen stock tube. Cell name, harvest/cell number, cryopreservation concentration, cryopreservation time are indicated on the cryopreservation tube.
(5) Resuscitation
Before operation, the experimental environment is checked, the equipment and reagent information is confirmed, and the required consumables and instruments are transferred into the sterile field of the cell preparation shop as specified. The super clean bench is opened for ventilation, the indicator lamp turns green, and the whole super clean bench is uniformly wiped by sterile gauze containing 75% alcohol, and required appliances, consumables and the like can be put into the super clean bench after being sterilized by 75% alcohol. And taking out the cell freezing tube from the liquid nitrogen storage tank, and transferring the cell freezing tube into a sterile area of a cell preparation workshop according to a specification after rapid recovery. Cells in the cryopreservation tube were collected in a centrifuge tube and counted. Cells were collected by centrifugation and the supernatant was discarded. Based on the counting results, cells were resuspended in complete medium and plated. The culture dish was marked with the harvest/cell number, date of resuscitation, algebra, and placed in an incubator.
Example 2: identification of hUC-MSCs
(1) Phenotypic identification of hUC-MSCs
Cell transfer of example 1 was takenGeneration to P4 generation of hUC-MSCs, 0.25% pancreatin digestion, centrifugation, and supernatant removal when cells grow to 80% confluency. PBS was resuspended to 100. Mu.l (density 1X 10) 7 cells/mL). The labeling 3 tubes, the labeling and the antibody adding are respectively as follows: blank tube, isotype control tube (MSCs positive isotype cocktail. Mu.l+ PE MSCs negative isotype control cocktail 20. Mu.L), CD105-PerCPCy5.5, CD73-APC, CD90-FITC, CD45-PE, CD34-PE, CD19-PE (MSCs positive cocktail. Mu.l+ PE MSCs negative cocktail 20 mu.L) were incubated at 4deg.C for 30min in the absence of light, PBS was washed 2 times (4deg.C, 350g,5 min), 400. Mu.L PBS was resuspended, FCM was used to detect the positive rate of mesenchymal stem cell index such as CD90, CD73, CD105 and hematopoietic index such as CD34, CD19, CD 45.
(2) hUC-MSCs multidirectional differentiation function detection
Taking P4 generation MSCs, inducing osteogenesis and adipose tissue differentiation in vitro, and detecting related indexes through morphology and gene level.
(3) hUC-MSCs induced differentiation
hUC-MSCs are mixed according to the proportion of 5 multiplied by 10 4 Inoculating 2mL of the total substrate in each six-hole plate, placing at 37 ℃ and 5% CO 2 Is cultured in an incubator of (a). When the cell fusion degree reaches 60% -70%, the old culture medium is sucked and removed, and 2 mL/hole hUC-MSCs osteogenesis induction/adipose tissue induction differentiation complete culture medium is added. After 2-4 weeks, the cells morphology and osteogenic/adipose tissue condition were changed once every 3 days and stained with alizarin red/oil red O.
The hUC-MSCs used in the present invention were identified to exhibit a fibroblast-like morphology and plastic adhesion, conforming to the minimum standards of MSCs defined by the International Society for Cytotheraphy (ISCT).
Example 3: preparation of human umbilical cord mesenchymal stem cells
The preparation method comprises the following steps:
before operation, the experimental environment is checked, the equipment and reagent information is confirmed, and the required consumables and instruments are transferred into the sterile field of the cell preparation shop as specified. The super clean bench is opened for ventilation, the indicator lamp turns green, and the whole super clean bench is uniformly wiped by sterile gauze containing 75% alcohol, and required appliances, consumables and the like can be put into the super clean bench after being sterilized by 75% alcohol. The cells of example 1 were removed from the incubator, placed in an ultra clean bench, and the culture medium in the dish was aspirated into a waste liquid jar. Each dish was washed with 0.9% physiological saline. Pancreatin was added to each dish and digested at 37 ℃. After the digestion of the cells in all dishes is completed, stop solution is added to stop the digestion, and the cells are filtered by a cell sieve. The cell suspension was transferred to a 50mL centrifuge tube, counted, and the supernatant was discarded by centrifugation. Cell preparation suspensions (physiological saline containing 1% human albumin) were prepared. And (3) adding the prepared cell preparation suspension to resuspend the cells, and transferring the cell suspension into a transfer bag. Sealing by a sealing machine, and placing the sealing machine at the temperature of 4 ℃ for standby.
Example 4: influence of human umbilical cord mesenchymal stem cells on animal model
1. Animal model
MRL/lpr mice (female, 4 weeks old, 20.86.+ -. 2.6 g) were purchased from Shanghai Slac laboratory animal company (license number: SCXK (Shanghai) 2017-0005). Mice were maintained in a specific pathogen-free environment. Approved by the institutional animal research ethics committee of affiliated hospitals in the university of Guangdong medical science (license number: AHGDMU-LAC-I (1) 2209-b 050).
2. hUC-MSCs transplanting dosage and design basis
Putting the generation P5-P7 hUC-MSCs into a 50mL centrifuge tube, centrifuging (1000 rpm,4 ℃ for 10 minutes), removing supernatant, adding 1-2 mL of sterile physiological saline for resuspension, taking 10 mu L of cell suspension for cell counting and cell activity rate measurement, wherein the cell activity rate is above 98%.
Female MRL/lpr lupus mice of 6 weeks of age were randomized into 4 groups (non-transplanted control group, and low, medium, and high dose hUC-MSCs-transplanted group) by intervention, as follows: (1) the time of transplantation was early (1 transplantation at week 8 and 10, respectively, with a volume of 300 μl each); (2) the transplantation dose was classified as low (LD, 0.4X10) 6 Cell/10 g), medium (MD, 0.8X10) 6 Cell/10 g), high (HD, 1.2X10) 6 Cells/10 g) 3 groups (calculated using general formula "logs=0.8762+0.698 logS", conversion factor of human to mouse 11.2), while the non-transplanted Control group (Control, ctrl; 300 μl of saline was injected), and the specific grouping conditions are shown in table 1. All mice were sacrificed at week 14 for further analysis (time line diagram see fig. 2A).
TABLE 1 MRL/lpr mice grouping
3. Urine protein detection
Urine samples were collected periodically to detect changes in urine protein levels: each mouse was placed individually in a metabolic cage overnight (12 hours), urine samples were collected, and total volume and urine collection time were recorded. After centrifugation (800 g,4 ℃,10 min), the urine sample supernatant was removed to a clean tube and immediately stored at-80 ℃.
Coomassie brilliant blue method for detecting urine protein concentration: thawing urine sample on ice, sequentially adding prepared BSA standard (1.5 mg/mL,1.0mg/mL,0.75mg/mL,0.5mg/mL,0.25mg/mL,0.125mg/mL,0.025mg/mL, water) and 4 μl of each sample to 96-well plate, adding Quick start TM 200 mu L of Bradford reagent and incubating for 5-15 min at normal temperature. The proteinuria concentration was measured at 570nm absorbance. Then, the total amount of urine protein was calculated from the urine concentration, urine amount and urine collection time for 24 hours.
4. Mice were sacrificed to obtain material
MRL/lpr mice were tested weekly for body weight and total body weight was determined prior to mice sacrifice. The mice were sacrificed 4 weeks after the last hUC-MSCs transplantation (i.e., 14 weeks of age). The anaesthetized mice are injected intraperitoneally with 1% sodium pentobarbital (9 mu L/g body weight) prepared by normal saline, the heart punctures and takes blood, the blood specimens are rapidly placed in EDTA anticoagulation tubes, the blood specimens are placed in the room temperature for 5 minutes, and after the blood specimens are placed in a standing state at room temperature, the blood specimens are centrifuged at a low temperature (3000 rpm,4 ℃ for 10 minutes), and the supernatant is taken and placed in an ultralow temperature refrigerator at-80 ℃ for long-term storage for standby. Mice were sacrificed and their spleens were taken for photographic weighing, and spleen index was expressed as spleen/body weight. And placing the weighed spleen, kidney and lymph node in pre-cooled normal saline, removing the envelope, transversely cutting 2 pieces of kidney, spleen or lymph node tissues with the thickness of 3mm, placing in 4% paraformaldehyde for fixation for 24 hours, embedding 1 piece of kidney, spleen or lymph node tissue as paraffin, and storing 1 piece of kidney, spleen or lymph node tissue as OCT frozen embedded tissue in an ultralow temperature refrigerator at-80 ℃, and preparing frozen slices in the later stage for immunofluorescence detection.
5. Detection of serum related indicators
(1) Mouse serum cytokine detection
Serum was sent to Guangzhou Convergence Biotechnology Co Ltd
MAP liquid phase protein chip kit (Millipore, billerica, mass., USA) detects serum inflammatory cytokine levels, including IFN-gamma, TNF-a, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-13.
(2) Detection of mouse serum anti-nucleic anti-ibody by Elisa kit
(1) The frozen serum was rewarmed on ice and the Mouse anti-nuclear antibody ELISA Kit (Alpha Diagnostic, USA) kit was opened and samples were 1: and 150 dilution. (2) 100. Mu.L of standard, sample and negative control were added to the predetermined wells, respectively, using 96-well plates in the ANA kit, sealed with sealing film, and incubated at room temperature for 60 minutes. (3) The plate was then back-off with 200 μl of 1× Working Wash Solution per well using a row gun and gently slapping the plate with hand to rinse, then the plate was back-off with liquid, and the 96 well plate was back-off with force on a paper towel for several times to ensure removal of liquid from the wells. The same procedure was repeated 3 more times. (4) To wells with standard, sample and negative control, 100 μl of Anti-Mouse Ig HRP was added, and sealed with sealing film and incubated for 30 minutes at room temperature. The plate was then back-off with 200 μl of 1× Working Wash Solution gently tapped for each well, and the plate was then back-off with the liquid and the 96-well plate was tapped on paper towel with force several times to ensure removal of the liquid from the wells. The same procedure was repeated for 4 more washes. (5) 100. Mu.L of TMB Substrate was added to each well and incubated for 15 minutes in the dark. The liquid in the well will start to turn blue. (6) 100 mu L of Stop Solution is added into each hole, and the mixture is blown and uniformly mixed. Detecting OD value at 450nm and OD value at 630nm by enzyme labeling instrument A well background. Finally, the net OD value (OD 450 -OD 630 ) And (3) making a standard curve, and calculating the titer of each sample anti-nuclear anti-ibody according to the standard curve.
(3) Detection of mouse serum anti-dsDNA anti-ibody by Elisa kit
(1) The frozen serum was rewarmed on ice and the Mouse anti-dsDNA (Alpha Diagnostic, USA) kit was opened and samples were 1: and 3000 dilution. (2) 100. Mu.L of standard, sample and negative control were added separately to the wells and sealed with sealing film using 96-well plates in dsDNA kit, and incubated at room temperature for 60 minutes. (3) The plate was then back-off with 200 μl of 1× Working Wash Solution per well using a row gun and gently slapping the plate with hand to rinse, then the plate was back-off with liquid, and the 96 well plate was back-off with force on a paper towel for several times to ensure removal of liquid from the wells. The same procedure was repeated 3 more times. (4) To wells with standard, sample and negative control, 100 μl of Anti-Mouse Ig HRP was added, and sealed with sealing film and incubated for 30 minutes at room temperature. The plate was then back-off with 200 μl of 1× Working Wash Solution gently tapped for each well, and the plate was then back-off with the liquid and the 96-well plate was tapped on paper towel with force several times to ensure removal of the liquid from the wells. The same procedure was repeated for 4 more washes. (5) 100. Mu.L of TMB Substrate was added to each well and incubated for 15 minutes in the dark. The liquid in the well will start to turn blue. (6) 100 mu L of Stop Solution is added into each hole, and the mixture is blown and uniformly mixed. The OD at 450nm and the OD at 630nm were measured with a microplate reader to normalize the well background. Finally, the net OD value (OD 450 -OD 630 ) A standard curve was made from which the titer of anti-dsDNA autoantibodies was calculated for each sample.
(4) Elisa kit for detecting mouse serum complement C3
(1) The frozen serum was rewarmed on ice, the Mouse C3 ELISA Kit (Alpha Diagnostic, USA) was opened and samples were subjected to 1 with 1 x Working Sample Diluent: diluted 35000. (2) 100. Mu.L of standard, positive control, sample and negative were prepared using 96-well plates in the C3 kitThe sex control was added to the predetermined wells separately and sealed with a sealing film and incubated at room temperature for 60 minutes. (3) The plate was then back-off with 200 μl of 1× Working Wash Solution per well using a row gun and gently slapping the plate with hand to rinse, then the plate was back-off with liquid, and the 96 well plate was back-off with force on a paper towel for several times to ensure removal of liquid from the wells. The same procedure was repeated 3 more times. (4) To wells with standard, sample and negative control, 100 μl of Anti-Mouse Ig HRP was added, and sealed with sealing film and incubated for 30 minutes at room temperature. The plate was then back-off with 200 μl of 1× Working Wash Solution gently tapped for each well, and the plate was then back-off with the liquid and the 96-well plate was tapped on paper towel with force several times to ensure removal of the liquid from the wells. The same procedure was repeated for 4 more washes. (5) 100. Mu.L of TMB Substrate was added to each well and incubated for 15 minutes in the dark. The liquid in the well will start to turn blue. (6) 100 mu L of Stop Solution is added into each hole, and the mixture is blown and uniformly mixed. The OD at 450nm and the OD at 630nm were measured with a microplate reader to normalize the well background. Finally, the net OD value (OD 450 -OD 630 ) A standard curve was made from which the concentration of each sample C3 was calculated.
6. Biochemical parameter determination of mice
Serum biochemical parameters (including albumin/globulin ratio (a/G ratio), glycocholate (CG), triglycerides (TG), total Bile Acid (TBA), etc.) were determined using an automatic analyzer (Cobas 8000, roche, switzerland).
7. Flow cytometry detection of MRL/lpr mice peripheral blood B cell subtype changes
(1) Preparation of peripheral blood PBMCs single cell suspensions: the supernatant was removed from the peripheral blood sample, and 10 volumes of 1×lysis buffer (BD Biosciences, USA) were added thereto, and incubated at room temperature for 15 minutes in the dark, thereby removing red blood cells. Then 4mL of PBS was added, centrifuged (350 g,4 ℃ C., 10 min) and the supernatant was removed, leaving about 100. Mu.L.
(2) Closing: anti-mouse CD16/32 antibody (Biolegend, USA) was used in an amount of 2. Mu.L (1.0. Mu.g)/100. Mu.L system and blocked from light at room temperature for 10min.
(3) Extracellular antibodies were incubated as shown in table 2.
TABLE 2 peripheral blood flow
(4) Fixing: each sample was added with 250. Mu. L Fixation Buffer (BD Biosciences, USA) and fixed at 4℃for 20 minutes. 4mL of PBS was added, centrifuged (350 g,4 ℃ C., 10 min), and the supernatant was aspirated off, leaving about 250. Mu.L of the supernatant on-line.
(5) Flow type B cell subgroup surface index and gate-circle scheme
Referring to the relevant literature, detection of mouse B cell subpopulations and cell surface markers were:
CD19 - CD138 + Plasma Cells (PC), CD19 + CD138 + Plasmablasts (PB), CD19 + CD138 - IgG1 + Memory B cell (IgG 1) + Memory B cells，IgG1 + MB)，CD19 + CD138 - IgG1 - Memory B cell (IgG 1) - Memory B cells，IgG1 - MB)。
Wherein, igG1 + MB and IgG1 - MB can be subdivided into 3 memory B cell subsets, respectively, as follows:
CD80 + PD-L2 + double Positive memory B cells (Double Positive MB, DP) - MB)，CD80 - PD-L2 + Single Positive (Single Positive MB, SP - MB)，CD80 - PD-L2 - Double Negative memory B cells (Double Negative MB, DN) - MB). A specific loop door scheme (see fig. 2A).
8. MRL/lpr mouse kidney function detection
(1) Serum creatinine detection
(1) Opening a creatinine (Cr) determination kit (Nanjing built C011-2-1), and respectively adding 6 mu L of a sample, a standard substance and a blank control into a 96-well plate; (2) 180 mu L of enzyme solution A is added to each well, the wells are incubated for 5 minutes at 37 ℃, and OD value A1 is measured at 546 nm; (3) 60 mu L of enzyme solution B is added into each hole, the mixture is incubated for 5 minutes at 37 ℃, and OD value A2 is measured at 546nm wavelength; (4) and calculating the Cr content according to the formula of the specification.
(2) Serum urea nitrogen detection
(1) Opening a urea nitrogen (BUN) test box (Nanjing build C013-2-1), and adding 2 mu L of blank control, standard substance and sample into a 96-well plate respectively; (2) 25 mu L of buffer enzyme solution is added into each hole respectively, and the mixture is accurately incubated for 10 minutes at 37 ℃ after being uniformly mixed; (3) 100 mu L of enzyme color reagent and 100 mu L of alkaline sodium hypochlorite are respectively added into each hole, the mixture is fully and uniformly mixed, and incubated for 10 minutes at 37 ℃, and OD value is measured at 540nm wavelength; (4) BUN content is calculated according to the formula of the specification.
9. MRL/lpr mice kidney, spleen, lymph node paraffin embedding
(1) Paraffin block making
(1) Drawing materials and fixing: cutting tissue blocks with the thickness of about 3mm, and rapidly placing the tissue blocks in 4% paraformaldehyde solution for fixing for 24 hours at the temperature of 4 ℃; (2) gradient alcohol dehydration: sequentially adding the tissue blocks into 75% alcohol for 1 hour, 85% alcohol for 30 minutes, 95% alcohol for 30 minutes multiplied by 2, and 100% alcohol for 30 minutes multiplied by 2; (3) and (3) transparency: immersing the dehydrated tissue block into xylene I for 10 minutes and xylene II for 15 minutes, wherein the total time is two times; (4) wax dipping: the tissue block is soaked in paraffin I solution at 60 ℃ for 30 minutes after the tissue block is sucked to dry the dimethylbenzene by using paper towel, and then soaked in paraffin II solution for 60 minutes; (5) embedding: placing the tissue block in an embedding mould, adding a proper amount of dissolved paraffin, lightly touching the tissue block with forceps to center the tissue block, then placing the tissue block on an ice bag, quickly covering a cover of the mould after the paraffin at the bottom layer is solidified, and adding a proper amount of paraffin to enable the tissue block to be embedded completely. And (3) standing the paraffin blocks at normal temperature, solidifying, putting the paraffin blocks into a refrigerator at the temperature of minus 20 ℃ for freezing for 20 minutes, taking the paraffin blocks out of the mold, and storing the paraffin blocks in the refrigerator at the temperature of minus 20 ℃ for standby.
(2) Paraffin section production
(1) Repairing: the intact morphology of the kidney, spleen or lymph node was first repaired using a 10 μm repair in repair mode. (2) Slicing: the slice mode was changed to the corresponding slice thickness, the kidney slice thickness was 1.5 μm, and the spleen and lymph node slice thickness was 3 μm. (3) Spreading, slicing and fishing: placing the cut paraffin tissue slices into warm water at 42 ℃ of a slice spreading machine for spreading. After the paraffin section tissue is fully unfolded, the continuous section is divided into a plurality of sections by using forceps, the sections are fished up by using a slide, and the slide is vertically placed on a slide frame for draining. (4) Baking slices: placing the slice tissue face up on a slice baking machine (60 ℃) to bake slices for 60-120 min, and preserving the slices in a refrigerator at 4 ℃ or minus 20 ℃ for standby after cooling.
10. Kidney pathology staining
(1) Kidney hematoxylin-eosin (H & E) staining procedure:
(1) dewaxing: xylene 10 min x 2; (2) hydration: 100% alcohol 10 s- & gt 95% alcohol 10 s- & gt water washing 30s; (3) nuclear dyeing: hematoxylin 6 min- & gt water washing 3 times, about 5 min- & gt 1% hydrochloric acid alcohol 2 s- & gt water washing 2 s- & gt 1% ammonia water 2 s- & gt water washing 2s; (4) dyeing cytoplasm: 1% eosin for 4 min, water washing for 1 min, 100% alcohol for 2s; (5) 100% alcohol 2 s- & gt baking, and sealing.
(2) Kidney periodate schiff (PAS) staining step:
(1) dewaxing: xylene 10 min x 2; (2) hydration: 100% alcohol 10 s- & gt 95% alcohol 10 s- & gt water washing 30s; (3) 1% of periodic acid for 25 minutes, and washing with water for 30 seconds; (4) PAS dye liquor 30 minutes- & gt soaking water 2 minutes; (5) nuclear dyeing: hematoxylin 6 min- & gt water washing 3 times, about 5 min- & gt 1% hydrochloric acid alcohol 2 s- & gt water washing 2 s- & gt 1% ammonia water 2 s- & gt water washing 2s; (6) dyeing cytoplasm: 1% eosin for 4 min- & gt water washing for 1 min; (7) 100% alcohol 2 s- & gt baking, and sealing.
(3) Kidney MASSON staining procedure:
(1) dewaxing: xylene 10 min x 2; (2) hydration: 100% alcohol 10 s- & gt 95% alcohol 10 s- & gt water washing 30s; (3) water bath of Bouin liquid at 65 ℃ for 30 minutes, and flushing with running water; (4) nuclear dyeing: hematoxylin 6 min- & gt water washing 3 times, about 5 min- & gt 1% hydrochloric acid alcohol 2 s- & gt water washing 2 s- & gt 1% ammonia water 2 s- & gt water washing 2s; (5) washing with 1% acetic acid washing solution for 1 time; (6) MASSON solution for 15 min (do not wash with water); (7) washing 3 times with 1% acetic acid washing liquid; (8) 1% bright green 25 s- & gt water washing 10s; (9) 100% alcohol 2 s- & gt baking, and sealing.
11. Immunofluorescence detection of renal IgG deposition
(1) Dewaxing and hydration
1.5 mu m paraffin sections were taken out from 4℃and baked at 60℃for 2h,xylene dewaxed 15min x 2 times after cooling. Sequentially hydrating ethanol with different concentrations: 100% ethanol 15min x 2 times; 95% ethanol 15min×2 times; 75% ethanol, ddH 2 O was 5min X1 times each.
(2) Microwave repair
Placing the slices into 1 XEDTA antigen retrieval liquid, placing into a microwave oven for microwave retrieval, boiling the liquid, and then placing the slices into high fire for 2min and medium high fire for 15min.
(3) 5% BSA blocking
ddH 2 O shaker wash for 5min and PBS solution of 0.1% Triton X-100 (background removed) for 20min, ddH 2 O-shaker wash 5min X3 times. The tissue periphery was wiped dry with paper towels and the pen was immunohistochemical circled. Mu.l of 5% BSA was added dropwise to each sample and incubated for 1h.
(4) Antibody incubation
Diluted direct standard antibody donkey anti-mouse IgG Alexa Fluor 647 (Cat#: A-21236) was formulated (1:400 dilution, 1% BSA dilution). Mu.l of primary antibody solution was added dropwise to each sample, and the mixture was allowed to stand at 4℃overnight. The PBS was washed 5min X3 times.
(5) DAPI (DAPI-nuclear stain)
DAPI stock solution PBS was diluted 5-fold and stained for 10min. The PBS was washed 5min X3 times.
(6) Sealing piece: and (5) sealing the anti-fluorescence quenching agent.
(7) Shooting: and shooting by a confocal fluorescent microscope, and randomly selecting 10-20 visual fields for each slice.
12. Results
(1) Ratio of hUC-MSCs viable cells isolated from different age precursors and intercepted different umbilical cord positions
To investigate the differences in isolated hUC-MSCs from different precursors and from different umbilical cord locations, the viability of hUC-MSCs was examined by flow cytometry and the results are shown in FIG. 1. It was found that the umbilical cord at the same location (umbilical cord cut at a location about 10cm from one end of the neonate (toward the mother) had higher viability of the hUC-MSCs cells derived from the 20-25 year old mother (fig. 1A-a) and significantly reduced mortality in mice after transplantation of the hUC-MSCs cells derived from the 20-25 year old mother, compared to the hUC-MSCs prepared from the maternal source (n=6) aged 30 years old; isolated hUC-MSCs cells from umbilical cord from the same parent (20-25 year old) (n=6) were significantly more active than isolated hUC-MSCs cells from umbilical cord from a position of about 10cm at one end of the neonate (maternal direction) were significantly less active (fig. 1B-a) than isolated hUC-MSCs cells from umbilical cord from a position of about 10cm at one end of the parent (maternal direction), and the mortality of mice after transplantation of isolated hUC-MSCs cells from umbilical cord from a position of about 10cm at one end of the neonate (maternal direction) was significantly reduced (fig. 1B-B). As described above, the activity of hUC-MSCs isolated from umbilical cords, which are 10cm long, at a position (toward the maternal direction) located at about 10cm from one end of the neonate, was higher when the maternal age was between 20 and 25 years, and the mortality of MRL/lpr mice was significantly reduced after transplantation.
(2) Early stage autoantibody level changes in MRL/lpr mice disease
To investigate the disease state of MRL/lpr mice prior to hUC-MSCs transplantation, serum anti-double-stranded DNA (anti-ds-DNA) and anti-nuclear antibody (ANA) levels of 6-week-old mice were detected using ELISA kits, with MRL/MPJ as a normal control, as shown in FIG. 2. As a result, it was found that the levels of anti-ds-DNA (a) antibodies and ANA (b) in serum of mice from the 6-week-old MRL/lpr lupus model were significantly higher than those of MRL/MPJ mice. This suggests abnormal levels of autoantibodies (anti-ds-DNA antibodies and ANA) at the early stage of lupus onset (i.e., 6 weeks of age).
(3) Effect of hUC-MSCs transplantation on mouse autoantibodies and spleen weight
In order to investigate the effect of hUC-MSCs transplantation on treatment of early lupus mice, ELISA was used to detect the MRL/lpr mouse serum antibody related index, and the results are shown in FIG. 3. The results showed that medium concentration of hUC-MSCs transplants (MD group) reduced anti-ds-DNA antibody levels (FIG. 3A), low concentration (LD group) and medium concentration (MD group) of hUC-MSCs transplants significantly reduced anti-nuclear antibody (ANA) levels (FIG. 3B). Mice were sacrificed at 14 weeks of age, the spleen weight ratio of the mice was monitored, spleen images are shown in fig. 3C, and spleen weights of mice in the medium-high concentration hic-MSCs transplanted group (MD group and HD group) were significantly reduced compared to the control group (fig. 3D). These results indicate that early hUC-MSCs transplantation has an inhibitory effect on spleen enlargement, which may be related to the inhibition of lymphocyte proliferation by hUC-MSCs.
(4) hUC-MSCs transplantation affects differentiation of mouse peripheral blood B cell subpopulations
The above experiments found that the transplantation of hUC-MSCs reduced spleen weight ratio in mice with early lupus, possibly associated with reduced lymphocyte proliferation. The results showed a significant increase in serum autoantibody (ANA and anti-ds-DNA antibodies) levels at the early stages of lupus onset, further detecting changes in B cell subsets following the engraftment of the hUC-MSCs. The surface or intracellular marker antibodies of the different B cell subsets were used for labeling and the changes of each B cell subset were analyzed by flow cytometry as shown in fig. 4. Lymphocytes were first gated, then single cell gated using FSC-A and FSC-H maps. After single cell gate, the total number of plasma cells (CD 19) was identified in the CD19 and CD138 maps - CD138 + Cell count and plasmablasts (CD 19) + CD138 + Cells). Next, igG1 can be recognized by using IgG1 + Plasma cell, igG1 - Plasma cell, igG1 + Plasma cell and IgG1 + Plasma cells. Furthermore, CD19 + CD138 - IgG1 + B cells can be identified as total igg1+ memory B cells. Three total IgG1 were further identified using CD80 and PD-L2 + Memory B cell subsets, respectively double positive memory B cells (DP MB, CD80 + PD-L2 + ) Single positive memory B cells (SP MB, CD 80) - PD-L2 + ) And double negative memory B cells (DN MB, CD 80) - PD-L2 - ) (FIG. 4A).
The data show that hUC-MSCs transplantation versus B cell population (including plasma cell population and plasmablasts population, C19 + CD138 - B cells) and Plasma Cell (PC) populations were unaffected, but medium-high concentration of hic-MSCs transplantation significantly reduced the total plasma blast Population (PB) (fig. 4B). Further B cell subtype analysis showed (FIG. 4C), MD and HD group IgG - PB cell (a) and IgG1 + The number of PB cells (b) was significantly lower than in the Ctrl group. However, hUC-MSCs transplantation was performed on IgG1 in peripheral blood of lupus mice - PC cell (c) and IgG1 + P cells (d) had no effect.
In addition, the number of memory B cell subtypes was determined by flow cytometry, as shown in fig. 4D. MD and HD group IgG compared to control group + Memory B (MB) cell numbers were significantly reduced (a). Further analysis of the B cell subtype showed that the hUC-MSCs transplantation significantly reduced the doubleNegative memory B cells (DN MB) and single positive memory B cells (SP MB) ratio, but no effect on double positive memory B cells (DP MB). As described above, hUC-MSCs can regulate proliferation and differentiation of B cells, affect production of inflammatory factors and antibodies, and thereby alleviate disease progression in lupus mice.
(5) hUC-MSCs transplantation can reduce serum inflammatory cytokine level
Serum samples were tested using protein chip technology. As a result, it was found that the serum inflammatory factors TNF-. Alpha.in the MD group (FIG. 5A), IFN-. Gamma.in the FIG. 5B, and IL-6 (FIG. 5C) were significantly lower than those in the control group, and that the HD group TNF-. Alpha.in the FIG. 5A, IL-6 (FIG. 5C), and IL-13 (FIG. 5D) were significantly lower than those in the control group. IFN-gamma levels were also significantly reduced compared to the control group compared to the LD group (FIG. 5B). However, hUC-MSCs transplanted had no effect on IL-1β (FIG. 5E), IL-2 (FIG. 5F), IL-4 (FIG. 5G) and IL-10 (FIG. 5H) levels. The result shows that the hUC-MSCs transplantation can relieve the serum inflammation environment of lupus mice and reduce the serum inflammation cytokine level.
(6) The hUC-MSCs transplantation can improve the kidney function of mice
Since lupus disease easily affects the kidney, kidney function was evaluated by detecting an index related to urine protein level or the like, and the results are shown in fig. 6. The 24 hour urine protein (a) and albumin/creatinine ratio (ACR) (b) were significantly lower in the MD and HD mice than in the control (fig. 6A). Serum creatinine (Scr) (a) and urea nitrogen (BUN) (B) levels were significantly reduced in the hiuc-MSCs transplanted group compared to the control group, and serum complement C3 (C) levels were higher in both the LD and MD groups than in the control group (fig. 6B). In addition, as shown in fig. 6C, serum biochemical index results of lupus mice were examined, the HD group had a higher a/G ratio (a) and CG (b) than the control group. MD group TG levels were significantly reduced compared to the control group (c). Serum TBA (d), inorganic phosphorus (P) (e) and UA (f) levels were significantly lower in the hUC-MSCs transplanted groups than in the control group. The result shows that the hUC-MSCs transplantation has a certain improvement effect on the kidney function and serum biochemical index of the lupus mice.
(7) The hUC-MSCs transplantation can improve kidney injury
In addition to the detection of kidney function related indexes, the kidneys of mice are collected to prepare paraffin. The protection of MSCs transplants in MRL/lpr mouse kidneys was observed and analyzed using H & E, PAS, masson pathology staining, and the results are shown in fig. 7. Pathological staining results showed that the inflammatory cell infiltration of glomeruli of mice transplanted with hUC-MSCs was significantly reduced, and glomerular fibrosis was significantly reduced (FIG. 7A). Immunofluorescence results showed significantly lower IgG and IgM antibody deposition in the kidneys of the MRL/lpr mice transplanted with hic-MSCs than in the control group (fig. 7B). The results show that the early lupus mice can reduce kidney injury by carrying out hUC-MSCs transplantation.
(8) hUC-MSCs inhibiting SLE patient peripheral B lymphocyte function
Peripheral blood was collected from SLE patients (approved by the ethical committee of hospitals, informed consent was obtained from the patients and family members) clinically diagnosed as light disorder and B lymphocytes were enriched and isolated. The results of co-culturing isolated B cells with hUC-MSCs at a ratio of 1:5, 1:1, 5:1 are shown in FIG. 8. The hUC-MSCs are found to significantly reduce CD138 + CD19 + The proportion of B cells (plasma cells) in the total enriched B cells significantly reduced the total IgG antibody level in the culture supernatant. Indicating that hUC-MSCs can inhibit B cell differentiation and reduce the capacity of B cells to produce antibodies.
As described above, the individual transplantation of hUC-MSCs into the light MRL/lpr mice can significantly inhibit the autoimmune reaction intensity, reduce the serum inflammatory factor level, improve the kidney pathology, protect the kidney function, and have excellent curative effects without side effects such as infection (figures 2-8).
In conclusion, low concentration (LD: 0.4X10) 6 Cell/10 g) and high concentrations of hoc-MSCs (HD: 1.2X10 6 Cell/10 g) transplantation showed a better dose-response relationship, whereas medium concentration (MD: 0.8X10 6 Cells/10 g) showed the best therapeutic effect. The hUC-MSCs prepared by the invention can effectively treat early (mild) systemic lupus erythematosus, can effectively inhibit the peripheral blood B cell differentiation of MRL/lpr mice (the B cell subgroup detected by the invention is relatively comprehensive), reduce the serum inflammatory factor level, improve the kidney function, alleviate kidney injury and reduce the generation of IgG total antibodies by the B cells of SLE patients. The umbilical cord mesenchymal stem cells prepared by the invention have the advantages different from the prior art: weak immunogenicity, and no immunological rejection; no further disputes concerning society, ethics, law; the source is sufficient; separating square The method is simple, the cell activity is strong, and the amplification is easy; umbilical cord mesenchymal stem cells are derived from human umbilical cord tissue and are a more primitive population of stem cells.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A preparation method of a human umbilical mesenchymal stem cell preparation, which is characterized by comprising the following steps:
(1) Obtaining primary human umbilical cord mesenchymal stem cells;
the primary human umbilical cord mesenchymal stem cells are from neonatal umbilical cord tissue produced by term caesarean section; the age of the mother of the newborn is 20-25; the umbilical cord tissue is an umbilical cord tissue with the length of 5-10 cm cut at the position of 8-12 cm at one end of the umbilical cord neonate;
(2) Culturing and amplifying the primary human umbilical cord mesenchymal stem cells to obtain passaged human umbilical cord mesenchymal stem cells;
(3) And preparing the cell suspension from the passaged human umbilical cord mesenchymal stem cells.
2. The method of claim 1, wherein in the step (1), the method of obtaining primary human umbilical cord mesenchymal stem cells comprises:
taking the umbilical cord tissue, cleaning and disinfecting, removing arteries and veins, and cutting the umbilical cord tissue into a surface area of about 10-25 mm 2 Standing for 15-30 min, adding culture solution, standing at 37deg.C in 5% CO 2 Culturing under the condition of the culture medium to obtain primary human umbilical cord mesenchymal stem cells.
3. The method of claim 1, wherein in the step (2), the method of culturing and expanding the primary human umbilical cord mesenchymal stem cells comprises:
taking the primary human umbilical cord mesenchymal stem cells, adding pancreatin, and digesting at 37 ℃ to obtain a cell suspension; centrifuging the obtained cell suspension, removing supernatant, adding fresh culture solution, resuspending cells, standing at 37deg.C and 5% CO 2 And (5) culturing.
4. The method of claim 1, wherein in step (3), the method of preparing a cell suspension comprises:
taking the passaged human umbilical cord mesenchymal stem cells, cleaning, adding pancreatin, digesting at 37 ℃, and filtering by a cell sieve to obtain a cell suspension; centrifuging the obtained cell suspension, removing supernatant, adding cell preparation suspension to resuspend cells, and packaging; the cell preparation suspension is physiological saline containing 1% of human albumin.
5. The method according to claim 1, wherein in the steps (1) to (3), the medium of the cells is MEM-alpha medium containing 5% serum replacement and has a pH of 7.2 to 7.4.
6. The method of claim 3 or 4, wherein the digestion is with PBS containing 0.25% trypsin without EDTA.
7. A human umbilical mesenchymal stem cell preparation prepared by the method of any one of claims 1 to 6.
8. The use of the preparation method of any one of claims 1 to 6, the human umbilical mesenchymal stem cell preparation of claim 7, in the preparation of a transplantation medicament for treating or preventing early or mild systemic lupus erythematosus.
9. The use according to claim 7, wherein the transplantation dosage of the transplantation drug is 0.4 x 10 6 ～1.2×10 6 Human umbilical cord mesenchymal stem cells/10 g; the infusion times are 1-3 times, and 200-300 mu L each time.
10. The preparation method of any one of claims 1 to 6, and the application of the human umbilical cord mesenchymal stem cell preparation of claim 7 in preparing medicines for co-culturing human umbilical cord mesenchymal stem cells and peripheral blood B cells of patients with early or mild systemic lupus erythematosus.
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