CN105769916B - Application of the source for mesenchymal stem cells excretion body in preparation treatment preeclampsia drug or preparation - Google Patents
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Abstract
The invention discloses application of the umbilical cord mesenchymal stem cells source excretion body in treatment preeclampsia preparation, and in particular to the excretion body treatment of preeclampsia of its generation is extracted with human umbilical cord mesenchymal stem cells.The excretion physical efficiency is effectively improved the symptom of preeclampsia hypertension and proteinuria, mitigate glomerulonephritis symptom, reduce the inflammatory reaction in placenta, and improve fetus development, and this excretion body also has the convenient advantage of storage and transport, this just provides new strategy for the treatment of the intractable inflammation related disease such as preeclampsia.
Description
Technical field
The present invention relates to the purposes of source for mesenchymal stem cells excretion body, specifically the excretion body of mescenchymal stem cell generation
Application in preeclampsia treatment.
Background technique
Preeclampsia (preeclampsia, PE) or be pre-eclampsia, be complication specific to the gestational period, feature
It is hypertension (systolic pressure >=140mmHg or diastolic pressure >=90mmHg) and albuminuria (>=300mg/ occur after pregnant woman's gestation 20 weeks
24 hours or random albuminuria >=++), it can be involved each organ of body and system.PE disease incidence accounts for the 3%~10% of all gestation,
It is the one of the major reasons for leading to pregnant and lying-in women and perinatal infants dead rate, the cause of disease does not illustrate yet, and clinically still lacks effective pre-
Anti- and treatment means.
Recent study shows that PE is a kind of arteriopathy inflammatory reaction of the mother to gestation, with low dosage lipopolysaccharides (LPS)
It can induce rat and mouse generate PE sample symptom.Immune imbalance may participate in the morbidity of PE, and PE patient's tire at Maternal-fetal interface
Disk include decidua, amnion, trophocyte source the dysfunction of mescenchymal stem cell (MSCs) may be PE patient Mu Tai circle
One of an important factor for face immune imbalance.Therefore, may be using the immunoregulation functional treatment PE of normal mesenchymal stem cell
A kind of new effective treatment means.We have confirmed that the mesenchyma in normal pregnancy pregnant woman's umbilical cord source is dry thin in the experiment of early period
Born of the same parents are injected intravenously the PE mouse model of I type T helper cell induction, can improve hypertension, albuminuria and the glomerulonephritis of mouse
Equal PE sample symptom, improves placenta development situation.
However, mescenchymal stem cell implant after there are the risk of malignant transformation and tumorigenesis, and the storage of cell and
Transport is inconvenient, limits clinical application.It is worth noting that MSCs can secrete a large amount of excretion body (exosome), outside these
The characteristics of secreting body is: diameter is between 30-150nm；Density is between 1.10-1.19g/mL；Expression specificity albumen, such as CD63
With Alix etc.；Carry the signal of interest molecule, including protein, lipid and RNA etc. of MSCs；It remains and parental cell
(MSCs) similar biological activity, the effects of such as adjusting immune response, go back to the nest to inflammation part and participate in tissue repair.Cause
This, the excretion body that MSCs is generated can be developed into the new method of cell-free treatment PE a kind of.
The report of the excretion body treatment PE in the source MSCs is had not yet to see, we for the first time generate people's umbilical cord source MSCs
Excretion body be applied to the treatment of PE, and achieve the good efficacy similar with MSCs.Compared with traditional cell therapy, excretion
Body has the advantages that stability is good, save and application is convenient and efficient.
Summary of the invention:
The present invention provides a kind of excretion body in human umbilical cord mesenchymal stem cells source in preparation treatment preeclampsia preparation
Application.Excretion body of the present invention derives from umbilical cord mesenchymal stem cells, and extracts by certain method, can be in -80 degree
Active (at least 1 year) is kept for a long time in refrigerator.
The excretion body is prepared as follows and obtains: by the umbilical cord mesenchymal stem cells culture supernatant of collection in 4 DEG C,
2,000g centrifugations 10 minutes, to remove dead cell and big fragment；Carefully supernatant is transferred in new sterile centrifugation tube, in
4 DEG C, 10,000g centrifugation 30 minutes, to remove organelle and little particle；Supernatant is carefully transferred to sterile ultracentrifugation pipe
In, in 4 DEG C, 110,000g ultracentrifugation 70 minutes, carefully discard supernatant, then plus the cleaning of injection physiological saline it is primary, in 4
DEG C, 110,000g ultracentrifugation 70 minutes, obtained precipitating is excretion body.Not according to the volume for the culture medium initially collected
Together, injection physiological saline is added as one sees fit to be resuspended, detects total protein concentration with Bradford kit, -80 DEG C of packing save.
The excretion body surface reaches significant PROTEIN C D63 and Alix, and average diameter is seen at 100nm or so, transmission electron microscope
The form observed all meets the feature of excretion body.
First passage zoopery of the present invention proves: the excretion body is similar with the effect of its mother cell, can effectively change
It is apt to the preeclampsia sample symptom of pregnant mouse, including reduces blood pressure and albuminuria, mitigate glomerulonephritis symptom, reduces the inflammation in placenta
Disease reaction improves fetus development.
The beneficial effects of the present invention are:
1. extracted umbilical cord mesenchymal stem cells source excretion physical efficiency is effectively improved preeclampsia hypertension and proteinuria
Symptom, mitigate glomerulonephritis symptom, reduce placenta in inflammatory reaction, and improve fetus development.This is preeclampsia
Treatment provides new approach.
2. excretion body provided by the invention can spend long-term preservation (at least 1 year) -80 compared with traditional cell therapy,
It can be used after dissolution, avoid the inconvenience that MSCs freezes and recovers, and may cause the wind of cell viability and function change
Danger.
3. mescenchymal stem cell excretion body raw material prepared by the present invention is umbilical cord, the acquisition of umbilical cord is non-invasive operation and category
It is recycled in waste, the problems such as not being related to Legal ethics, umbilical cord quantity is sufficient, from a wealth of sources, can carry out large-scale production.
Detailed description of the invention:
The marker protein CD63 and Alix of Fig. 1 Western-blot method detection source MSCs excretion body；
The form of Fig. 2 transmission electron microscope observing excretion body: scale 100nm.
The influence of Fig. 3 MSCs and its excretion body to the pregnant mouse blood pressure of PE sample: compared with the control group,#p<0.05；Treatment group with
Model group ratio,*p<0.05。
The influence of Fig. 4 MSCs and its excretion body to the pregnant mouse Urine proteins of PE sample: compared with the control group,#p<0.05；Treatment group
With model group ratio,*p<0.05。
The influence that 1 MSCs of table and its excretion body develop the pregnant mouse placenta of PE sample and fetus.
Fig. 5 MSCs and its excretion body change the pregnant mouse renal tissues pathology of PE sample: HE stained slice figure, scale 50
Macrophages infiltration situation in Fig. 6 placenta tissue: immunohistochemistry figure, the CD68 positive is macrophage.
The influence of Fig. 7 MSCs and its excretion body to inflammatory factor level in placenta tissue, compared with the control group,#p<
0.05；Treatment group and model group ratio,*p<0.05。
Specific embodiment:
Embodiment 1 is separately cultured human umbilical cord mesenchymal stem cells (MSCs), and (reagent consumptive material used in laboratory passes through nothing
Bacterium processing)
Multipara's informed consent acquires the umbilical cord of normal full-term pregnancy Cesarean esction fetus.Before umbilical cord acquisition puerpera need through
Cross stringent pathogen detection, including microspironema pallidum, AIDS virus, cytomegalovirus, hepatitis B, hepatitis C virus, plum
The microorganisms such as poison and mycoplasma, use after confirming safety.
The nearly placenta end umbilical cord 20-30cm of clip is placed in the sterile phosphate buffer (PBS) of 4 DEG C of pre-coolings and saves, and 4 is small
When interior use.In superclean bench, umbilical cord is cut into the segment of 5cm or so, the blood of umbilical cord surface residual is cleaned with PBS；
After removing arteria umbilicalis and umbilical vein, it is placed in and is cultivated containing the DMEM/F12 of 0.01% penicillin and streptomysin (being purchased from Sigma company)
In base (being purchased from Gibco company, similarly hereinafter), 2mm is cut into surgical scissors3The tissue block of left and right；The tissue shredded is transferred to
In 50ml centrifuge tube, 4 DEG C, 300g centrifugation carefully discard upper layer culture medium, are added and organize isometric mixture slaking enzyme, set
In 37 DEG C of shaking tables, 200rpm oscillation digests 1.5-2 hours；Wherein mixture slaking enzyme (is purchased from containing II Collagenase Type 2mg/ml
Sigma company), neutral proteinase 0.8mg/ml (is purchased from Amresco company), and hyaluronidase 0.03mg/ml (is purchased from Sigma
Company), it is configured with DMEM/F12 culture medium.Postdigestive tissue fluid is centrifuged 10 minutes in 4 DEG C, 400g, is carefully discarded supernatant；
It is washed twice with PBS, 4 DEG C every time, 400g centrifugation 10 minutes abandon supernatant；Finally precipitating is resuspended in containing (the purchase of 12% fetal calf serum
In DMEM/F12 culture medium in Gibco company, similarly hereinafter), spread into T-25 culture bottle (Corning company), in 37 DEG C, 5%CO2
It is cultivated in incubator.After 3 days, not adherent cell and tissue are discarded, the fresh DMEM/ containing 10% fetal calf serum is added
F12 culture medium.When cell length to 80% degrees of fusion (about 7 days), digested with 0.25% pancreatin (being purchased from Gibco company), passage
Continue amplification cultivation to 10 centimetres of diameter of culture dishes (Corning company, similarly hereinafter) are middle, and is denoted as 1st generation.Later every three to four
It changes secondary culture after liquid or digestion, and every passage is once denoted as a generation.
The raw material that mescenchymal stem cell is obtained in the present embodiment is umbilical cord, and the acquisition of umbilical cord is non-invasive operation and belongs to useless
The problems such as gurry recycles, and is not related to Legal ethics, umbilical cord quantity is sufficient, from a wealth of sources, can carry out large-scale production.
The extraction and identification of 2 source MSCs excretion body of embodiment
(1) the extraction of the source MSCs excretion body
The MSCs culture supernatant for collecting 3-7 generation (is purchased from 500ml sterile centrifuge bottles or 50ml polypropylene centrifuge tube
Beckman company), it is centrifuged 10 minutes in 4 DEG C, 2000g, to remove dead cell and big fragment.Carefully supernatant is transferred to
In new sterile centrifugation tube, it is centrifuged 30 minutes in 4 DEG C, 10,000g, to remove organelle and little particle.Carefully supernatant is turned
It moves on in sterile ultracentrifugation pipe, in 4 DEG C, 110,000g (Beckman ultracentrifuge) ultracentrifugation 70 minutes, carefully discards
Supernatant, then plus the cleaning of injection physiological saline it is primary, in 4 DEG C, 110,000g ultracentrifugation 70 minutes, obtained precipitating was outer
Secrete body.Different according to the volume for the culture medium initially collected, the injection physiological saline that small size is added as one sees fit is resuspended, and uses
After Bradford kit (being purchased from Thermo company) detects total protein concentration, -80 DEG C of packing are saved.
(2) the identification of the source MSCs excretion body
With the significant PROTEIN C D63 and Alix of western-blot method analysis excretion body: suitable protein lysate being added to split
Solve excretion body precipitating, abundant whirlpool concussion；After measuring protein concentration, 20 μ g total proteins are taken, add 5 × SDS sample-loading buffer, 99
It is heated 10-15 minutes in DEG C metal bath；12,000g is centrifuged 5 minutes；Loading is 10%SDS-PAGE；100V transferring film 70 minutes,
5% skim milk is closed 1 hour；The primary antibody for being separately added into anti-CD 63 and Alix (is diluted by 1:500 with TBS, is purchased from Santa
Cruz company), 4 DEG C of overnight incubations；TBST is washed film 5 minutes × 4 times, adds corresponding secondary antibody, is incubated at room temperature 1.5 hours；TBST washes film 5
Minute × 4 times, add ECL luminescent solution (being purchased from Millipore company), is taken pictures by the imaging of chemiluminescence gel imaging system.Knot
Fruit is as shown in Figure 1, CD63 and Alix positive expression in the excretion body in the source MSCs, and the expression both in cell is very
Low, this is consistent with document report.
With the form of transmission electron microscope observing excretion body: excretion body being mixed well, the load sample that 20 μ l drip to diameter 2mm is drawn
On copper mesh, it is stored at room temperature 5 minutes, carefully sops up surplus liquid with filter paper.It is added dropwise uranium acetate negative staining 2 minutes, is inhaled with filter paper
Fall surplus liquid, is dried under incandescent lamp；Transmission electron microscope 80-120kv is imaged and takes pictures.As a result as shown in Figure 2, it is seen that diameter is
The rounded sac balloon-shaped structure of 100nm or so, meets the feature of excretion body.
Embodiment result explanation, the excretion body in the source MSCs, this excretion body surface can be successfully obtained with differential centrifugation
Up to significant PROTEIN C D63 and Alix, the form that average diameter is observed at 100nm or so, transmission electron microscope all meets excretion body
Feature.It is worth noting that, being reported according to existing literature, excretion body can be saved in -80 degree, and the activity of holding at least 1 year is not
Become, can be used after dissolution.Avoiding problems the inconvenience that conventional cell treatment MSCs used freezes and recovers, and thus band
The risk that the cell viability and function come changes.
The therapeutic effect of 3 MSCs of embodiment and its excretion body to preeclampsia (PE) mouse
C57/B6 mouse 8-10 weeks used, it is purchased from model animal research institute, Nanjing University.
(1) low dosage lipopolysaccharides (LPS) inducing mouse PE model
Mouse is mated in female and the ratio of male 2:1, and check vaginal plug in the morning, and having vaginal plug, person is considered becoming pregnant into
Function, is denoted as gestation the 0.5th day, and so on.Pregnant mouse is randomly divided into 4 groups: control group, model group, MSC treatment group and MSC come
Source excretion body treatment group, every group 8.Referring to method (the Tiziana Cotechini et of Tiziana etc.
al.Inflammation in rat pregnancy inhibits spiral artery remodeling leading to
fetal growth restriction and features of preeclampsia.J Exp Med.2014,211(1):
165-179), PE sample symptom is generated with low dosage LPS inducing mouse.Model group: from the 13.5 to 16.5th day, every morning abdominal cavity
The LPS (model 0111:B4, similarly hereinafter, Sigma company) of injecting normal saline dissolution, injection dosage were 10 μ g/ at the 13.5th day
Kg, following three days are 40 μ g/kg.MSC treatment group: lps injection time and the same model group of method, in addition respectively at the 14.5th
It and the 16.5th day tail vein injection 0.5 × 106MSCs (is resuspended in 100 μ l physiological saline).Excretion body treatment group: LPS note
Time and the same model group of method are penetrated, in addition (is resuspended in respectively at the 14.5th day and the 16.5th day 15 μ g excretion body of tail vein injection
In 100 μ l physiological saline).Control group: the physiological saline of injection equal volume daily.
(2) mouse blood pressure and Urine proteins detection
At gestation the 17.5th day, using the tail systolic pressure of tail sleeve method measurement mouse, as a result as shown in figure 3, normal control
The pregnant mouse systolic pressure of group is 98 ± 4.8mmHg, and the systolic pressure of the pregnant mouse of model group of LPS induction is increased to 116 ± 6.9mmHg, and right
According to group compared to statistical significance (p < 0.05)；And the systolic pressure of MSCs and its excretion body intervention group is respectively 102.7 ± 4.4
And 101.1 ± 6.1mmHg, there is significant difference (p < 0.05) compared with model group.
It is deprived of food but not water in pregnant 16.5th day evening to pregnant mouse, collects pregnant mouse urine with compressing bladder method after 12 hours.
Collected urine, which is collected by centrifugation after supernatant, to be placed on -80 DEG C of refrigerators and freezes in case detection.The detection of albuminuria is according to the white egg of mouse
The specification of white enzyme-linked immunosorbent assay quantification kit (bethyl company) carries out.As a result as shown in figure 4, Normal group
It compares, the urine protein level of the pregnant mouse of model group of LPS induction is significantly raised (p < 0.05)；And MSCs and its pre- equal energy of excretion soma
The urine protein level for significantly reducing the pregnant mouse of PE sample has significant difference (p < 0.05) compared with model group.
(3) placenta and the observation of fetus development condition
At gestation the 17.5th day, cervical dislocation put to death pregnant mouse, and pregnant mouse placenta, uterus and fetus are won in dissection immediately, are counted
Litter size and implantation number weigh the weight of every placenta and fetus.The results are shown in Table 1, compared with Normal group, LPS
The average every pregnant mouse placental weight of the model group of induction reduces, and the fetus number in bosom is reduced, fetus weight loss, implantation number mesh
Increase；And MSCs and its excretion soma is pre- equal can improve the pregnant mouse placenta of PE sample and the hypogenetic situation of fetus.
The influence that table 1.MSCs and its excretion body develop the pregnant mouse placenta of PE sample and fetus
Note: model group and control group ratio,#p<0.05；Treatment group and model group ratio,*p<0.05。
(4) kidney and placenta tissue pathological examination
Paraffin embedding after the fresh renal tissue won is fixed overnight with 4% paraformaldehyde, slice, then routinely step
Carry out HE (haematoxylin Yihong) dyeing.As a result as shown in figure 5, compared with Normal group, the renal glomerulus base of LPS induction group
Counterdie thickens, and proliferation of mesangial cells, extracellular matrix increases；And MSCs and its excretion soma can be relieved the change of these pathology in advance
Change.Placenta tissue does immunohistochemistry, observes the macrophage quantity of the CD68 positive in tissue.As shown in fig. 6, and Normal group
It compares, macrophages infiltration increases in the model group placenta tissue of LPS induction；And MSCs and its excretion soma it is pre- can be reduced it is huge
Phagocyte is infiltrated to placenta tissue.
(5) placenta tissue inflammatory factor detects
The RNA for extracting placenta tissue, does quantitative fluorescent PCR (polymerase chain reaction) after reverse transcription, detects inflammatory factor
TNF-α, the mRNA (mRNA) of IL-1 β and IL-6 are horizontal, using GAPDH as internal reference.The primer: mouse TNF-α forward direction is drawn
Object: CCCTCACACTCAGATCATCTTCT, reverse primer: GCTACGACGTGGGCTACAG；Mouse IL-1 β forward primer:
GAAATGCCACCTTTTGACAGTG, reverse primer: TGGATGCTCTCATCAGGACAG；Mouse IL-6 forward primer:
TGCCTTCTTGGGACTGAT, reverse primer: TAAGCCTCCGACTTGTGA；Mouse GAPDH forward primer:
AACGACCCCTTCATTGAC, reverse primer: TCCACGACATACTCAGCAC.Each gene C t value that PCR reacts uses
2-△CtMethod analysis target gene relative expression quantity.As a result as shown in fig. 7, compared with Normal group, the mould of LPS induction
Inflammatory factor TNF-α in type group placenta tissue, IL-1 β and IL-6 expression significantly increase；And MSCs and its excretion soma
The pre- expression that can significantly reduce these inflammatory factors in placenta.
Above embodiments result explanation: the excretion body of source for mesenchymal stem cells is similar with the effect of its mother cell, can have
Effect improves the preeclampsia sample symptom of pregnant mouse, therefore has considerable application value in preparation treatment preeclampsia preparation.
Statistical analysis
Statistical data is provided in the form of mean+SD, and with 5 software of GraphPad Prism, (U.S. Holy Land is sub-
Brother) it is for statistical analysis and map, two groups are compared using t-test, and p < 0.05 is with significant difference.
Claims (3)
1. application of the excretion body of source for mesenchymal stem cells in preparation treatment preeclampsia drug or preparation, feature exist
In the excretion body of the source for mesenchymal stem cells by being effectively improved the symptom of preeclampsia hypertension and proteinuria, mitigate kidney
Bead ephritis symptom reduces the inflammatory reaction in placenta, and improves fetus development and achieve the purpose that treat preeclampsia, thus real
The application in preeclampsia drug or preparation is treated in preparation now；The excretion body of the source for mesenchymal stem cells reduces placenta
The expression of middle inflammatory factor TNF-α, IL-1 β and IL-6, and then mitigate glomerulonephritis symptom and reduce in placenta
Inflammatory reaction；The mescenchymal stem cell is umbilical cord mesenchymal stem cells.
2. application according to claim 1, which is characterized in that the excretion body, which is dissolved in injection physiological saline, to be made
It is used at injection.
3. application according to claim 2, which is characterized in that the administration mode of the excretion body is intravenous injection.
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