CN109666748A - The evaluation method of umbilical cord mesenchymal stem cells biological efficacy in terms for the treatment of hepatopathy - Google Patents
The evaluation method of umbilical cord mesenchymal stem cells biological efficacy in terms for the treatment of hepatopathy Download PDFInfo
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- CN109666748A CN109666748A CN201910102838.4A CN201910102838A CN109666748A CN 109666748 A CN109666748 A CN 109666748A CN 201910102838 A CN201910102838 A CN 201910102838A CN 109666748 A CN109666748 A CN 109666748A
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Abstract
The invention discloses the evaluation methods of umbilical cord mesenchymal stem cells biological efficacy in terms for the treatment of hepatopathy, and when the difference that the Ct value that the mRNA level in-site Ct value of the 3rd representative up to hepatocyte growth factor of human umbilical cord mesenchymal stem cells subtracts reference gene GAPDH obtains is greater than 7.95, the human umbilical cord mesenchymal stem cells in the 3rd generation of judgement biological efficacy in terms for the treatment of hepatopathy is unqualified.It is easy using sample process when detection method detection of the invention, using the RNA extracts kit of commercialization, detection can be completed within 2 hours.
Description
Technical field
The present invention relates to the gene expression selective mechanisms technologies of Diseases of Hematopoietic Stem Cell specificity orientation application, specifically, relating to
And a kind of detection method of umbilical cord mesenchymal stem cells treatment hepatopathy aspect biological efficacy.
Background technique
Umbilical cord mesenchymal stem cells (umbilical cord-mesenchymal stem cells, UC-MSCs) are from navel
With a kind of adult stem cell with multi-lineage potential and multiple biological function isolated in tissue.
Waste of the umbilical cord after fetus production, the acquisition of umbilical cord has natural advantage, because of umbilical cord and placenta
Biohazard Waste, and the wound not additional to contributor's (donor), the not puzzlement of Medical Ethics are belonged in China.With
Human body others tissue site such as marrow and fat etc. are compared, and MSCs is obtained from umbilical cord has multiple advantages, including source is rich
Richness, MSCs ratio is high, the proliferative capacity of MSCs is strong, lower immunogenicity, stronger immunosuppression capability, donor is not present
Wound etc..MSCs can secrete more vegetative growth factors, such as hepatocyte growth factor etc., be very beneficial for MSCs performance
The function of histoorgan reparation.
Verified hepatocyte growth factor (HGF) is the most strong cellular factor for promoting hepatocyte growth and liver regeneration.
HGF stimulates the proliferation of liver ancestral cells to repair hepatic injury.HGF can also cause the strong expression of Bcl-xL, block Fas and its
Signal transduction after ligand activation, and then the hepatocellular apoptosis for inhibiting Fas to mediate, to treat burst liver failure caused by Fas
It exhausts.HGF is other than the rapid proliferation that can induce hepatic parenchymal cells, moreover it is possible to stimulate bile duct epithelial cell to be proliferated, thus influence entire
The development of organ.Since HGF has the characteristic for promoting hepatocyte growth and inhibiting apoptosis, so that HGF may be hepatopathy cell therapy
A key factor.We and other research groups have been found that the HGF of MSCs secreting high levels.Due to umbilical cord source
MSCs secretes higher HGF than the MSCs of derived from bone marrow, and it is the slow extra urgaent dispatch liver of MSCs clinical treatment hepatitis B that this, which can be explained equally,
Failure, but the effect of umbilical cord MSCs treatment is substantially better than the therapeutic effect of bone marrow MSCs, hence it is evident that improve the life of hepatic failure patients
Deposit rate.In addition, research discovery MSCs has the function of diminishing inflammation while inhibiting immune response.Therefore, MSCs has weight
The research and application prospect wanted especially all have important clinical value in terms for the treatment of hepatopathy disease.
Currently, the definition to only one popularity of MSCs, i.e., the mesenchyma that 2006 international cell therapy associations propose
Three standards that stem cell defines, and lack and treat functional definition, it cannot judge that the MSCs by culture is more properly directed to
Certain a kind of disease, also lacks the judgement of disease associated biological efficacy.With the intensification of research, different are gradually recognized
The MSCs in people source is likely that there are point of the power for the treatment of functionally, also lacks effectively and efficiently has targetedly biological
Learn effect detection means.Therefore, deficiency in order to overcome the above problems, we are to the MSCs in umbilical cord source in hepatocyte growth factor
Sub (HGF) gene expression layer has carried out system research, by large sample detection come analysis and summary comparison data, it was demonstrated that the table of HGF
It reaches, in Different Individual kind, there are apparent differences, and also there is different difference in terms of different culture algebra.Accordingly,
We have proposed the new methods that a kind of detection MSCs is screened in the gene expression level of hepatopathy specificity orientation application.
Summary of the invention
The biology effect that the technical problem to be solved by the present invention is to overcome existing mescenchymal stem cells in terms for the treatment of hepatopathy
The deficiency of power detection means establishes the new method of a set of effective detection umbilical cord mesenchymal stem cells treatment hepatopathy potential.
The present invention provides a kind of detection methods of umbilical cord mesenchymal stem cells biological efficacy, it is characterised in that passes through inspection
The expression quantity of the HGF-mRNA of umbilical cord mesenchymal stem cells is surveyed to judge that it treats the effect of hepatopathy.
In a specific embodiment, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and
Its judgment criteria are as follows: real time quantitative PCR method detects the expression quantity of the HGF gene mRNA of human umbilical cord mesenchymal stem cells with difference
It is threshold value for 7.95 (differences=CtHGF-CtGAPDH), the judgement higher than this numerical value is that human umbilical cord mesenchymal stem cells treat liver
The effect of disease is unqualified.Calculation formula used in the present invention, i.e. difference=CtHGF-CtGAPDH are a Δ Ct method, because only
It needs the Ct value of HGF to compare with the reference gene (GAPDH) of oneself, obtains the expression of the HGF of this crowd of MSCs relative to interior
Join the difference of the expression of gene.Expressing gene due to not being related to HGF compares with other expressing genes, also not
The expressing gene for being related to the HGF of MSCs before and after the processing compares, therefore the present invention is without Δ Ct method twice.
The present invention use for the first time multisample detect different cell culture algebra umbilical cord MSC HGF-mNRA, pass through statistics
Method, using SPSS software, calculate the MSCs in 32 different umbilical cords sources P3 for when HGF mRNA relative expression quantity water
95% flat confidence interval.The higher the better by the HGF secreted due to MSCs, because this invention uses under 95% confidence interval
Limit value is threshold value, is lower than this threshold value, then is judged as that this crowd of MSCs is not suitable for being used to treat hepatopathy.
The beneficial effects obtained by the present invention are as follows being: it is easy using sample process when detection method detection of the invention, it utilizes
The RNA extracts kit of commercialization can complete detection within 2 hours.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the MSC in 32 different umbilical cord sources, the mRNA of HGF compared relatively with the mRNA of internal reference GAPDH after data；
Fig. 2 is 6 samples in the umbilical cord MSC for randomly select 32 separate sources, then continues culture to P20 from P3 generation
Generation, P3, P6, P10, P15, P20 for when, respectively continue real time quantitative PCR method detection MSCs HGF relative expression quantity.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings, it should be understood that preferred reality described herein
Apply example only for the purpose of illustrating and explaining the present invention and is not intended to limit the present invention.
Embodiment
The extraction and reverse of total serum IgE are cDNA
32 umbilical cords are had collected altogether, MSC is isolated from umbilical cord, after from originally culture to the third generation, in super-clean bench ring
It is digested under border, then uses PBS centrifuge washing 2 times, mentioned with the RNA extracts kit (product of Invitrogen company) of commercialization
The total serum IgE of MSCs is taken out, Nanodrop detects the 260/280OD value of total serum IgE.According to OD value, Reverse Transcriptase kit is utilized
(product of Promega company) is cDNA segment mRNA reverse transcription.
The mRNA relative expression quantity of real-time quantitative PCR detection HGF
| Primer | Sequence |
| GAPDH-S | 5'TCGACAGTCAGCCGCATCT 3' |
| GAPDH-A | 5'CCGTTGACTCCGACCTTCA 3' |
| HGF-S | 5'AGTGGCATCAAATGTCAGCC 3' |
| HGF-A | 5'GCTTGTGAAACACCAGGGTC 3' |
Real-time PCR is 7500 models of ABI company, and consumptive material and reagent are purchased from ABI company.
After real-time quantitative PCR detects, the MSCs of 32 samples sources P3 for when, HGF gene and GAPDH gene
Detected value (Ct value) is as follows.
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| GAPDH | 16.241 | 15.818 | 15.62 | 16.761 | 16.462 | 15.313 | 14.985 | 15.649 | 15.803 | 15.945 |
| HGF | 23.586 | 24.07 | 23.5 | 25.011 | 26.401 | 23.393 | 22.648 | 22.978 | 22.975 | 23.263 |
| Number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| GAPDH | 15.333 | 14.681 | 16.004 | 14.922 | 15.171 | 15.213 | 14.94 | 15.477 | 15.023 | 14.661 |
| HGF | 22.919 | 22.325 | 23.382 | 21.782 | 22.709 | 22.659 | 22.208 | 23.604 | 21.761 | 23.445 |
| Number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| GAPDH | 15.268 | 14.903 | 14.25 | 15.353 | 14.895 | 15.925 | 13.971 | 14.114 | 14.614 | 15.47 |
| HGF | 24.006 | 23.333 | 20.875 | 22.892 | 21.367 | 22.313 | 22.064 | 22.427 | 22.021 | 22.515 |
| Number | 31 | 32 | ||||||||
| GAPDH | 14.604 | 15.065 | ||||||||
| HGF | 22.855 | 22.127 |
The processing of data and statistical analysis
According to the comparative approach of gene expression amount, the relative expression quantity using Δ differential technique, that is, gene is fixed in real time
The Ct value that amount PCR instrument detected subtracts the Ct value of reference gene, i.e. Δ value=CtHGF-CtGAPDH.
Therefore, the P3 of 32 samples is respectively as follows: for the Ct value of MSCs
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Δ value | 7.345 | 8.252 | 7.88 | 8.25 | 9.939 | 8.08 | 7.663 | 7.329 | 7.172 | 7.318 |
| Number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Δ value | 7.586 | 7.644 | 7.378 | 6.86 | 7.538 | 7.446 | 7.268 | 8.127 | 6.738 | 8.784 |
| Number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Δ value | 8.738 | 8.43 | 6.625 | 7.539 | 6.472 | 6.388 | 8.093 | 8.313 | 7.407 | 7.045 |
| Number | 31 | 32 | ||||||||
| Δ value | 8.251 | 7.062 |
These data are inputted statistic software SPSS 17.0,95% confidence interval is calculated, it is as follows to obtain result:
Case processing abstract
Description
These data are inputted statistic software SPSS 17.0,95% confidence interval is calculated, it is as follows to obtain result:
Since the higher the better for the relative expression quantity of HGF, this discovery uses the lower limit value of 95% confidence interval as threshold
Value, i.e., 7.9511, take 2 significant digits, as 7.95.
Fig. 1 is that the MSC in 32 different umbilical cords sources obtains one after the mRNA of HGF is compared relatively with the mRNA of internal reference GAPDH
Group contains the data of 32 numerical value, then carries out average evaluation and calculates standard deviation to get the schematic diagram of Fig. 1 is arrived.
Fig. 2: randomly selecting 6 samples in the umbilical cord MSC of 32 separate sources, then continues culture to P20 from P3 generation
Generation, P3, P6, P10, P15, P20 for when, respectively continue real time quantitative PCR method detection MSCs HGF relative expression quantity.From
In P10 generation, starts, and the expression quantity rapid decrease (numerical value is higher, and practical expression quantity is lower) of HGF, statistical analysis shows this decline
Difference have statistical significance.When this prompt clinical application people umbilical cord MSCs treatment hepatopathy, avoid selection P6 for after as far as possible
Cell.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (4)
1. the evaluation method of umbilical cord mesenchymal stem cells biological efficacy in terms for the treatment of hepatopathy, which is characterized in that pass through detection
Umbilical cord mesenchymal stem cells express the mRNA level in-site of hepatocyte growth factor to evaluate the ability that it treats hepatopathy.
2. the evaluation method of umbilical cord mesenchymal stem cells as described in claim 1 biological efficacy in terms for the treatment of hepatopathy,
It is characterized in that, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and the cell algebra of detection was the 3rd generation.
3. the evaluation method of umbilical cord mesenchymal stem cells according to claim 2 biological efficacy in terms for the treatment of hepatopathy,
It is characterized in that, when the mRNA level in-site Ct value of human umbilical cord mesenchymal stem cells expression hepatocyte growth factor subtracts reference gene
The difference that the Ct value of GAPDH obtains determines that the human umbilical cord mesenchymal stem cells in the 3rd generation are raw in terms for the treatment of hepatopathy when being greater than 7.95
Object effect is unqualified.
4. the evaluation method of umbilical cord mesenchymal stem cells biological efficacy according to claim 1-3, feature
It is, uses the mRNA level in-site of real-time quantitative PCR detection umbilical cord mesenchymal stem cells expression hepatocyte growth factor.
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