CN112430667A - Method for evaluating treatment effect of mesenchymal stem cells in Parkinson's disease - Google Patents
Method for evaluating treatment effect of mesenchymal stem cells in Parkinson's disease Download PDFInfo
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Abstract
The invention relates to a method for evaluating the treatment effect of mesenchymal stem cells on Parkinson's disease, which specifically comprises the following steps: the method comprises the following steps of PD model construction, rotation behavior monitoring, UC-MSCs tail vein transplantation treatment, tissue drawing, immunohistochemistry, fluorescence quantitative Q-PCR, dopamine content detection, Superoxide Dismutase (SOD) activity and Malondialdehyde (MDA) content detection. The invention has scientific and reasonable design and high and stable effect, provides a method for evaluating the treatment effect of Parkinson's disease, selects 6-OHDA as a modeling agent, has the modeling dose of 10 mu g, selects a proper modeling site AP: +0.7mm, MR: -3.0mm and DV: -5.0mm from a former fontanelle reference point, adopts a brain stereotaxic technology, is a simple, high-efficiency and stable PD rat model, has the success rate of more than 80 percent, and provides experimental basis and theoretical basis for establishing a PD treatment strategy and designing a novel PD treatment medicament.
Description
Technical Field
The invention belongs to the field of biological medicines, relates to a Parkinson disease treatment technology, and particularly relates to an evaluation method of a Parkinson disease treatment effect by using mesenchymal stem cells.
Background
The brain is the material basis of consciousness and sensation of human and higher animals, the consciousness of human and animals is closely related to perfect brain function, the behavior of the brain is governed by brain activity, the damage of a part of the brain necessarily causes the attenuation of corresponding function, and the abnormal metabolism of neurotransmitter can cause various neurological diseases, such as Alzheimer disease, Parkinson disease and the like. Parkinson's Disease (PD), also known as paralysis agitans, is a frequent extrapyramidal disease in the middle-aged and elderly, one of the most common neurodegenerative diseases.
Epidemiological shows that more than 1% of people in China have PD diseases, men are more susceptible than women, the main symptoms of the PD diseases are static tremor, slow movement, autonomic nervous dysfunction, myotonia, abnormal gait of posture, cognitive/mental abnormality, sensory disturbance and the like, the main pathology of the PD diseases is changed into degeneration and necrosis of nigral Dopamine (DA) neurons, the striatal DA content is obviously reduced, Tyrosine lightening enzyme (TH) is a marker protein of the DA neurons in brain, the DA biosynthesis is regulated, and the content change of the Tyrosine lightening enzyme is closely related to the occurrence of the PD diseases.
The PD disease has various pathogenic factors, and may be related to age, genetic factors, social factors, drug factors, oxidative stress, apoptosis, damage of ubiquitin-protease system, abnormal protein deposition and other factors. PD disease is an incurable disease at present, drugs such as dopamine receptors and monoamine oxidases can only relieve PD disease symptoms, and the action of the drugs is gradually weakened or even some complications appear along with the progress of the disease course, so that the pathogenesis, clinical behavior change and pathological biochemical change of the drugs are deeply researched, an efficient modeling method is researched, a disease animal model is built, and a suitable therapeutic drug is inevitably searched.
At present, chemical reagents are mainly used for constructing a PD animal model in a scientific research PD model, wherein the main reagent is 6-hydroxyl-dopamine (6-OHDA), the 6-OHDA is a high-efficiency poison for rats and primates and is widely used for manufacturing unilateral injury models, the most common modeling method is to inject the 6-OHDA to substantia nigra or central forebrain tracts in a unilateral mode or to inject the 6-OHDA directly into striatum bodies, the former enables drugs to be accumulated in DA neurons and generate toxicity to the DA neurons, the DA neurons can die quickly in a short time and are similar to an acute MPTP model, the latter enables the neurons to be injured slowly in four weeks and is mainly used for simulating the chronic course of PD and further enabling the DA neurons to generate degenerative changes, and the two models have the main advantages that the PD animal model can be monitored by rotational behavioral visual quantification, therefore, the method is often used for observing the effect of the drug on dopaminergic nerves in pharmacological and pharmacodynamic researches.
Stem Cells (SC) are a type of pluripotent cells with self-replication ability, and can be differentiated into various tissue cells under in vivo or in vitro specific induction conditions, wherein umbilical cord mesenchymal stem cells (UC-MSC) derived from healthy umbilical cord tissues are one of important members in a stem cell family, have strong proliferation ability and multidirectional differentiation potential, can be used for immunoregulation, have the advantages of being wide in application due to the fact that materials are available, moral and ethical disputed, large in the number of available cells, strong in fertility, high in stem cell characteristics after multiple passage amplification, safe and reliable in use, simple in preparation and the like, and have obvious advantages in treatment of tissue injury, diabetes, aging diseases and neurodegenerative diseases.
The UC-MSCs selected in the invention can effectively improve the immunity of an organism, obviously improve the behavior of a PD rat, repair damaged neurons of the brain, and obviously improve the antioxidant index of the organism, and has the advantages of low cost, sufficient umbilical cord source, easy separation, expansion and culture and strong multiplication capacity, and the UC-MSCs used as a cell transplantation material can be suitable for treating clinically common PD diseases.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for evaluating the treatment effect of mesenchymal stem cells applied to Parkinson's disease, establishes a stable PD rat model, has a success rate of over 60 percent, seeks a safe and effective neuron protective drug, improves PD rat behaviours through UC-MSCs transplantation treatment, repairs PD rat dopaminergic neurons, is used for increasing the number of TH + cells of PD rats, increasing the dopamine content in PD rat brain striatum and reducing oxidative stress reaction, and realizes the research of UC-MSCs on treating Parkinson's disease.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a method for evaluating the treatment effect of mesenchymal stem cells on Parkinson's disease specifically comprises the following steps:
construction of a PD model
Selecting an SPF-grade SD rat, carrying out intraperitoneal injection of 2% pentobarbital for anesthesia, selecting coordinate positioning by taking bregma as a standard reference point, injecting 6-OHDA into the MFB and the right VTA area of the rat, slowly injecting 6-OHDA4 mu l at each point by a Hamilton micro injector, wherein the injection time is 5min, keeping a needle for 10min, and carrying out postoperative intraperitoneal injection of 5 ten thousand U of penicillin;
the monitoring of the rotational behavior is prepared
Performing intraperitoneal injection of 0.5mg/kg apomorphine for one week after brain stereotaxic modeling, continuously monitoring for 8 weeks, monitoring each rat for 30min each time, recording the total rotation number of the rat within 30min, wherein the total rotation number is more than 7r/min on average, and the rotation standard of the PD rat model is met, i.e. the Parkinson's PD rat model is successfully established;
trigonopsis hystrix UC-MSCs tail vein transplantation treatment
Tissue sampling
Firstly, taking whole brain tissue after UC-MSCs transplantation treatment is finished;
② after the transplantation treatment of UC-MSCs, directly taking fresh brain tissue;
fifthly immunohistochemistry
Cutting whole brain tissue at a thickness of-4.00 mm to-7.00 mm by taking bregma as a reference through brain map search, and carrying out section staining on the whole brain tissue to observe to obtain an immunohistochemical result;
sixthly, detecting relative expression quantity of the substantia nigra compact part TH by QRT-PCR
Taking 50 mg of frozen midbrain tissue directly from fresh brain tissue, extracting RNA, and measuring OD values at wavelengths of 260nm and 280nm respectively;
synthesizing CDNA according to the operation of a reverse transcription kit specification, and carrying out data statistics and analysis after finishing photographing every time according to the cycle of (1) 95 ℃ for 30s, (95) 95 ℃ for 30s, (50) 30s, (72) 30s and (39-fourth);
detecting dopamine content, SOD activity and MDA content.
In the construction of the PD model, 2 [ mu ] g, 5 [ mu ] g, 10 [ mu ] g, 15 [ mu ] g and 20 [ mu ] g of 5 gradients are designed in total by 6-OHDA, and the selected coordinate points are as follows:
furthermore, the preferred injection amount of 6-OHDA is 10. mu.g, and the preferred injection sites are AP: +0.7mm, MR: -3.0mm, DV: -4.5 mm.
Furthermore, the UC-MSCs tail vein transplantation therapy comprises the following method steps:
separating and culturing UC-MSC cells:
obtaining sterile umbilical cord tissue, peeling adventitia of the umbilical cord tissue and artery and vein in the umbilical cord tissue under sterile condition, separating umbilical cord Wharton's jelly, and shearing to 1-5mm3And is adhered to T150cm at a density of 2-3mm2Adding a proper amount of DMEM/HAM' SF-12+ 10% FBS culture solution into the bottom surface of the culture bottle for culture for 1-4 h, digesting the UC-MSCs by using 0.05% trypsin when the coverage rate of the UC-MSCs reaches about 80%, centrifuging and collecting the UC-MSCs, and counting the cell viability at 8000-10000 cells/cm according to the state of the cultured cells and the cell count viability of the collected cells2Transferring to a new culture bottle for continuous culture;
② preparing UC-MSCs cell preparation:
when the cells are cultured to the P3 generation and the cell coverage rate reaches 85 percent, digesting the cells, centrifugally collecting the cells at 300-400 g/min for 5-10 min, counting the cells, and adjusting the cell concentration to 1 x 10 by using normal saline6cells/ml, SD rat tail vein transplantation injection 500 u L with 500g weight;
③ treatment of caudal vein transplantation of UC-MSCs:
the UC-MSCs are administrated by adopting a tail vein slow bolus injection mode, the bolus injection speed is controlled to be 10s/100 mu L, and the administration volume is determined according to the body weight of a rat.
Further, the section staining method includes:
placing the cut brain tissue in 50%, 60%, 70%, 80%, 90%, 100% I and 100% II gradient alcohol in sequence, wherein each gradient is 40min, xylene I and xylene II are respectively 30min, paraffin I1h and paraffin II are subjected to paraffin embedding after overnight, cutting the tissue into slices with the thickness of about 4-6 mu m by using a paraffin slicer, and performing immunofluorescence detection;
② using 3% H2O2Soaking for 20min, inactivating endogenous peroxidase, washing with PBS for 5min × 3 times, soaking slices in 0.01M, PH 6.0.0 citrate buffer solution, heating to boil in a microwave oven, powering off, repeating for 1-2 times at an interval of 5-10 min, cooling, washing with PBS for 5min, incubating for 2-3 times, incubating at 37 ℃ with 5% BSA (bovine serum albumin) blocking solution for 30min, spin-drying, dropwise adding mouse anti-human TH polyclonal antibody, and incubating for 60min at 37 ℃;
③ 5min washing with PBS, 3 times, dripping secondary antibody: labeling goat anti-rabbit IgG with biotin, incubating at 37 ℃ for 30min, washing with PBS for 5min, 3 times, dropwise adding SABC, and incubating at 37 ℃ for 30 min;
rinsing with PBS for 5min × 3 times, dripping DAB as a color developing agent, rinsing with PBS for 3min × 3 times, counterstaining with hematoxylin for 1min, rinsing with PBS, respectively rinsing with 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, xylene I and xylene II for 1min, sealing with neutral gum, and air drying;
taking a picture by a fluorescence inverted microscope to obtain an immunohistochemical result.
Furthermore, a 25. mu.l reaction system was set up to perform the QRT-PCR experiment according to the following operating table:
| name (R) | Dosage (mu l) |
| SYBR | 12.5 |
| Primer F | 0.5 |
| Primer R | 0.5 |
| Form panel | |
| ddH2O | Up to 25μl |
。
And detecting the dopamine content, the SOD activity and the MDA content by using striatum tissue suspension, taking 40 mg of frozen striatum tissue, homogenizing by using a tissue homogenizer, cracking the tissue, preparing 10% homogenate according to the proportion of 1g to 9ml of physiological saline after full cracking, centrifuging for 20min at 3000-4000 rmp/min, sucking supernatant, determining the protein concentration by using a Coomassie brilliant blue method, and subpackaging for later use.
The method for extracting whole brain tissue includes: performing intraperitoneal injection on a rat by using 2% and 0.15 mg/kg of pentobarbital, fully anaesthetizing, fixing the rat on an anatomical plate, sequentially opening an abdominal cavity, fastening all organs with liquid circulation effects below the mediastinum and the chest by using hemostatic forceps, opening the chest, completely exposing the heart, inserting an aortic arch from the left ventricle by using a 7# perfusion needle, rapidly cutting off the right auricle, perfusing 150ml of physiological saline, allowing the blood to become clear, whitening the liver, perfusing and fixing by using PH7.4 and 4% paraformaldehyde by about 100ml, dissecting the brain in time after fixing is completed, taking out brain tissues, fixing in the paraformaldehyde of 4% and PH7.4, and standing by.
Furthermore, the method for directly taking fresh brain tissue comprises the following steps: intraperitoneal injection is carried out on 2% and 0.15 mg/kg of pentobarbital, after full anesthesia, the neck is quickly cut off, the brain is taken out, rat midbrain and striatum tissues are separated on ice, weighed and frozen at-80 ℃ for later use.
The invention has the advantages and positive effects that:
1. the invention provides a method for evaluating the treatment effect of mesenchymal stem cells on Parkinson disease, and relates to a method for treating Parkinson disease by using mesenchymal stem cells and the effect of the method, wherein 6-OHDA is selected as a modeling agent, the modeling dose is 10 mu g, a proper modeling site AP: +0.7mm, MR: -3.0mm and DV: -5.0mm are selected as a former fontanelle reference point, and a PD rat model is successfully constructed by a brain stereotaxic technique.
2. The invention carries out UC-MSCs tail vein transplantation treatment on a PD model mouse for 8 courses of UC-MSCs by a tail vein transplantation method after the model is successfully established, the UC-MSCs dose in each course of treatment is 1 multiplied by 106cells/Kg, and provides an optimal treatment dose of the protective action of umbilical cord mesenchymal stem cells on rat neurons.
3. The invention provides an optimal modeling dose and an optimal modeling site, a simple, efficient and stable PD rat model is established, the success rate reaches more than 80%, the detection of symbolic indexes shows that the PD model rat treated by UC-MSCs tail vein transplantation can be obviously improved in behavior compared with the untreated model rat PD rat, the phenomena of hair color and arch back are relieved, the dopaminergic neuron repair of the PD rat is obvious, the TH + cell number of the PD rat is increased, the dopamine content in the brain striatum of the PD rat is increased, the oxidative stress reaction is reduced, and the symptoms of the PD model rat are obviously relieved.
4. The invention has scientific and reasonable design and high and stable effect, provides a method for evaluating the treatment effect of Parkinson's disease, selects 6-OHDA as a modeling agent, has the modeling dose of 10 mu g, selects a proper modeling site AP: +0.7mm, MR: -3.0mm and DV: -5.0mm from a former fontanelle reference point, adopts a brain stereotaxic technology, is a simple, high-efficiency and stable PD rat model, has the success rate of more than 80 percent, and provides experimental basis and theoretical basis for establishing a PD treatment strategy and designing a novel PD treatment medicament.
Drawings
FIG. 1 shows the improvement effect of rotation behavior in the present invention;
FIG. 2 shows TH in substantia nigra of rats of various groups in the present invention+Results of changes in cell number and neuron number;
FIG. 3 shows TH in substantia nigra of rats of various groups in the present invention+The ratio of cell damage to normal side;
FIG. 4 shows the results of the variation of TH relative fluorescence intensity in brain tissue of rats in each group according to the present invention;
FIG. 5 shows the variation of Dopamine (DA) content in striatal tissues of rats in various groups according to the present invention;
FIG. 6 shows the results of the activity of superoxide dismutase (SOD) in striatal tissues of rats in various groups according to the present invention;
FIG. 7 shows the results of the variation of Malondialdehyde (MDA) content in striatal tissues of rats in various groups according to the invention.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The invention provides a method for evaluating the treatment effect of mesenchymal stem cells on Parkinson's disease, which specifically comprises the following steps: the method comprises the following specific operation steps of PD model construction, rotation behavior monitoring, UC-MSCs tail vein transplantation treatment, tissue drawing, immunohistochemistry, fluorescent quantitative Q-PCR, dopamine content detection, Superoxide Dismutase (SOD) activity and Malondialdehyde (MDA) content detection:
(1) construction of PD model
Selecting an SPF-grade SD rat with the weight of 180-220 g, fasting for 12h before model building, carrying out anesthesia by injecting 2% pentobarbital into an abdominal cavity, fixing the SD rat on a stereotaxic platform after sufficient anesthesia, disinfecting the brain skin by a conventional method, incising the skin of an operation area, scraping periosteum, positioning by using a brain stereotaxic instrument, selecting a coordinate to position by taking a bregma as a standard reference point, injecting 6-OHDA into an MFB and a right VTA area of the rat, slowly injecting 6-OHDA4 mu l into each point by a Hamilton micro injector, keeping a needle for 10min, slowly withdrawing the needle, suturing the brain skin, injecting 5 mu of penicillin into the abdominal cavity after operation, preventing postoperative infection, and observing postoperative recovery conditions every day.
In the experiment, 5 gradients of 2 mug, 5 mug, 10 mug, 15 mug and 20 mug are designed in total for 6-OHDA, and the coordinate points are selected as shown in the following table:
the optimal injection amount of 6-OHDA is determined by experiments to be 10 mug, the optimal injection site is AP: +0.7mm, MR: -3.0mm, DV: -4.5 mm.
(2) Monitoring of rotational behaviours
Injecting apomorphine (0.5mg/kg) into the abdominal cavity one week after brain stereotaxic modeling, inducing the rat to rotate towards the healthy side, slightly bending the back of the rat when rotating, having no obvious stress point on the injured side hind paw, continuously monitoring for 8 weeks, monitoring for 30min for each rat every time, recording the total rotation number of the rat within 30min, being more than average 7r/min, meeting the rotation standard of a PD rat model, and indicating that the Parkinson's PD rat model is successfully established.
(3) UC-MSCs tail vein transplantation treatment
And (3) isolated culture of UC-MSC cells: obtaining sterile umbilical cord tissue, peeling adventitia of the umbilical cord tissue and artery and vein in the umbilical cord tissue under sterile condition, separating umbilical cord Wharton's jelly, and shearing to 1-5mm3And is adhered to T150cm at a density of 2-3mm2After culturing for 1-4 h on the bottom surface of the culture bottle, adding a proper amount of DMEM/HAM' SF-12+ 10% FBS culture solution into the culture bottle for culturing; when the coverage rate of UC-MSCs reaches about 80%, digesting the UC-MSCs by adopting 0.05% trypsin, centrifugally collecting the UC-MSCs, and performing cell counting and survival rate of collected cells at 8000-10000 cells/cm according to the state of cultured cells2Transferring to a new culture flask, and continuing to culture.
Preparing UC-MSCs cell preparation: culturing until the cell coverage rate reaches about 85% when the cells are cultured for P3 generation, digesting the cells, centrifuging and collecting the cells at 300-400 g/min for 5-10 min, counting the cells, and adjusting the cell concentration to 1 x 10 by using normal saline6SD rats with cells/ml and 500g weight were injected with 500. mu.L of the tail vein graft.
Tail vein transplantation treatment of UC-MSCs: the UC-MSCs are administrated by adopting a tail vein slow bolus injection mode, the bolus injection speed is controlled to be about 10s/100 mu L, and the administration volume is determined according to the body weight of a rat.
(4) Tissue sampling
After transplantation treatment of UC-MSCs is finished, abdominal cavity injection is carried out on each group of rats by using 2% pentobarbital (0.15 mg/kg), the rats are fully anesthetized and fixed on a rat dissection board, the abdominal cavity is opened in sequence, hemostatic forceps are used for clamping the mediastinum and all organs below the chest, which play a role in liquid circulation, the chest cavity is opened, the heart is completely exposed, a 7# perfusion needle is used for inserting the aortic arch from the left ventricle, the right auricle is cut off rapidly, about 150ml of physiological saline is perfused, the blood which flows out becomes clear, the liver becomes white, the rats are perfused and fixed by using about 100ml of 4% paraformaldehyde (PH7.4), the brain is dissected timely after fixation is finished, and brain tissues are taken out and fixed in the 4% paraformaldehyde (PH 7.4.
Each group of rats was injected intraperitoneally with 2% pentobarbital (0.15 mg/kg), after sufficient anesthesia, the brains were removed by promptly severing the neck, rat midbrain and striatal tissue were isolated on ice, weighed and frozen at-80 ℃ for future use.
(5) Immunohistochemistry
Through brain atlas search, the expression level of the nerve Tyrosine Hydroxylase (TH) black brain pars compacta at the position of-4.44 mm to-6.60 mm (about 2mm thick) of the brain is determined to be more based on bregma, and brain tissues at the position of-4.00 mm to-7.00 mm are cut and stained.
According to the selected position, the cut brain tissue is sequentially placed in 50%, 60%, 70%, 80%, 90%, 100% I and 100% II gradient alcohol, each gradient is 40min, xylene I and xylene II are respectively 30min, paraffin I1h is adopted, paraffin II is used for paraffin embedding after overnight, a paraffin slicer is used for cutting the tissue into slices with the thickness of about 4-6 mu m, and immunofluorescence detection is carried out; with 3% H2O2Soaking for 20min to inactivate endogenous peroxidase; washing with PBS for 5min × 3 times, soaking the slices in 0.01M citrate buffer solution (pH6.0), heating with a microwave oven until boiling, powering off, repeating for 1-2 times at an interval of 5-10 min; after cooling, washing with PBS for 5min x (2-3) times, and incubating with 5% BSA blocking solution at 37 ℃ for 30 min; spin-drying, dripping mouse anti-human TH polyclonal antibody, and incubating at 37 deg.C for 60 min; washing with PBS for 5min × 3 times, adding dropwise secondary antibody (biotin-labeled goat anti-rabbit IgG), and incubating at 37 deg.C for 30 min; washing with PBS for 5min × 3 times, adding SABC dropwise, and incubating at 37 deg.C for 30 min; washing with PBS for 5min × 3 times, dripping DAB as color reagent, washing with PBS for 3min × 3 times, counterstaining with hematoxylin for 1min, washing with PBS, washing with 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, xylene I, and xylene II for 1min respectively, sealing with neutral gum, and air drying; and (4) taking a picture by a fluorescence inverted microscope to obtain an immunohistochemical result.
(6) QRT-PCR detection of relative expression quantity of TH (TH) of substantia nigra compacta
Taking 50 mg of midbrain tissue frozen at about, adding 1ml of Trizol, homogenizing by a tissue homogenizer, cracking the tissue, fully cracking, standing on ice for 5min, 12000rpm/min, centrifuging at 4 ℃ for 10min, separating, taking out supernatant, transferring into an EP tube treated by DEPC, adding 200 μ l of chloroform, covering a centrifugal tube cover tightly, vortexing and mixing fully, standing on ice for 5min, 12000rpm/min, centrifuging at 4 ℃ for 15min, dividing the mixture into 3 layers, taking RNA as the upper layer, taking DNA as the middle white membrane layer and protein as the lower layer, carefully absorbing the uppermost layer of liquid, putting into the EP tube treated by DEPC, adding isopropanol with the same volume, fully mixing, standing at room temperature for 10min, 10000rpm/min, centrifuging at 4 ℃ for 10min, discarding supernatant, slowly adding 1ml of 75% ethanol along the tube wall, slowly inverting up and down for 30s, 7500rpm/min, centrifuging at 4 ℃ for 5min, repeating the previous steps, removing the ethanol, fully drying, and adding a proper amount of DEPC water to dissolve the precipitate; and (3) quantitatively analyzing the concentration of the extracted RNA by using an enzyme-labeling instrument, determining OD values at wavelengths of 260nm and 280nm respectively, wherein the 260/280nm ratio of all measured samples is 1.8-2.0, so that the extracted RNA is free from pollution of protein and other impurities, and the experimental requirements are met.
Following the protocol of the reverse transcription kit (Taraka), CDNA was synthesized and QRT-PCR experiments were performed by establishing a 25. mu.l reaction system according to the following protocol.
| Name (R) | Dosage (mu l) |
| SYBR | 12.5 |
| Primer F | 0.5 |
| Primer R | 0.5 |
| Form panel | |
| ddH2O | Up to 25μl |
The method comprises the steps of circulating for 39 times at 95 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 30s and two to four, and taking a picture after each time is finished.
After the detection is completed, data statistics and analysis are carried out.
(7) Detection of dopamine content, SOD activity and MDA content
Detecting dopamine content, SOD activity and MDA content by using striatum tissue suspension, taking 40 mg of striatum tissue, performing homogenate by a tissue homogenizer, cracking the tissue, preparing 10% homogenate according to the proportion of 1g to 9ml of physiological saline after full cracking, centrifuging for 20min at 3000-4000 rmp/min, sucking supernatant, determining protein concentration by a Coomassie brilliant blue method, and subpackaging for later use.
The detection method comprises the steps of operating by adopting an enzyme-linked immunosorbent assay (ELISA) detection kit of Shanghai enzyme-linked biotechnology, namely, Limited, sequentially measuring the absorbance OD value of each hole under the condition of the wavelength of 450nm, and calculating the content of dopamine, the activity of SOD and the content of MDA.
The results of the experiment are shown in FIGS. 1 to 7.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (9)
1. A method for evaluating the treatment effect of mesenchymal stem cells in Parkinson's disease is characterized by comprising the following steps: the method specifically comprises the following steps:
construction of a PD model
Selecting an SPF-grade SD rat, carrying out intraperitoneal injection of 2% pentobarbital for anesthesia, selecting coordinate positioning by taking bregma as a standard reference point, injecting 6-OHDA into the MFB and the right VTA area of the rat, slowly injecting 6-OHDA4 mu l at each point by a Hamilton micro injector, wherein the injection time is 5min, keeping a needle for 10min, and carrying out postoperative intraperitoneal injection of 5 ten thousand U of penicillin;
the monitoring of the rotational behavior is prepared
Performing intraperitoneal injection of 0.5mg/kg apomorphine for one week after brain stereotaxic modeling, continuously monitoring for 8 weeks, monitoring each rat for 30min each time, recording the total rotation number of the rat within 30min, wherein the total rotation number is more than 7r/min on average, and the rotation standard of the PD rat model is met, i.e. the Parkinson's PD rat model is successfully established;
trigonopsis hystrix UC-MSCs tail vein transplantation treatment
Tissue sampling
Firstly, taking whole brain tissue after UC-MSCs transplantation treatment is finished;
② after the transplantation treatment of UC-MSCs, directly taking fresh brain tissue;
fifthly immunohistochemistry
Cutting whole brain tissue at a thickness of-4.00 mm to-7.00 mm by taking bregma as a reference through brain map search, and carrying out section staining on the whole brain tissue to observe to obtain an immunohistochemical result;
sixthly, detecting relative expression quantity of the substantia nigra compact part TH by QRT-PCR
Taking 50 mg of frozen midbrain tissue directly from fresh brain tissue, extracting RNA, and measuring OD values at wavelengths of 260nm and 280nm respectively;
synthesizing CDNA according to the operation of a reverse transcription kit specification, and carrying out data statistics and analysis after finishing photographing every time according to the cycle of (1) 95 ℃ for 30s, (95) 95 ℃ for 30s, (50) 30s, (72) 30s and (39-fourth);
detecting dopamine content, SOD activity and MDA content.
2. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: in the construction of the PD model, 2 mu g, 5 mu g, 10 mu g, 15 mu g and 20 mu g of 5 gradients are designed in total by 6-OHDA, and selected coordinate points are as follows:
3. the method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 2, wherein the method comprises the following steps: the preferred injection amount of 6-OHDA is 10. mu.g, and the preferred injection site is AP: +0.7mm, MR: -3.0mm, DV: -4.5 mm.
4. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: the UC-MSCs tail vein transplantation treatment method comprises the following steps:
separating and culturing UC-MSC cells:
obtaining sterile umbilical cord tissue, peeling adventitia of the umbilical cord tissue and artery and vein in the umbilical cord tissue under sterile condition, separating umbilical cord Wharton's jelly, and shearing to 1-5mm3And is adhered to T150cm at a density of 2-3mm2Adding a proper amount of DMEM/HAM' SF-12+ 10% FBS culture solution into the bottom surface of the culture bottle for culture for 1-4 h, digesting the UC-MSCs by using 0.05% trypsin when the coverage rate of the UC-MSCs reaches about 80%, centrifuging and collecting the UC-MSCs, and counting the cell viability at 8000-10000 cells/cm according to the state of the cultured cells and the cell count viability of the collected cells2Transferring to a new culture bottle for continuous culture;
② preparing UC-MSCs cell preparation:
when the cells are cultured to the P3 generation and the cell coverage rate reaches 85 percent, digesting the cells, centrifugally collecting the cells at 300-400 g/min for 5-10 min, counting the cells, and adjusting the cell concentration to 1 x 10 by using normal saline6cells/ml, SD rat tail vein transplantation injection 500 u L with 500g weight;
③ treatment of caudal vein transplantation of UC-MSCs:
the UC-MSCs are administrated by adopting a tail vein slow bolus injection mode, the bolus injection speed is controlled to be 10s/100 mu L, and the administration volume is determined according to the body weight of a rat.
5. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: the section staining method comprises the following steps:
placing the cut brain tissue in 50%, 60%, 70%, 80%, 90%, 100% I and 100% II gradient alcohol in sequence, wherein each gradient is 40min, xylene I and xylene II are respectively 30min, paraffin I1h and paraffin II are subjected to paraffin embedding after overnight, cutting the tissue into slices with the thickness of about 4-6 mu m by using a paraffin slicer, and performing immunofluorescence detection;
② using 3% H2O2Soaking for 20min, inactivating endogenous peroxidase, washing with PBS for 5min × 3 times, soaking slices in 0.01M, PH 6.0.0 citrate buffer solution, heating to boil in a microwave oven, powering off, repeating for 1-2 times at an interval of 5-10 min, cooling, washing with PBS for 5min, incubating for 2-3 times, incubating at 37 ℃ with 5% BSA (bovine serum albumin) blocking solution for 30min, spin-drying, dropwise adding mouse anti-human TH polyclonal antibody, and incubating for 60min at 37 ℃;
③ 5min washing with PBS, 3 times, dripping secondary antibody: labeling goat anti-rabbit IgG with biotin, incubating at 37 ℃ for 30min, washing with PBS for 5min, 3 times, dropwise adding SABC, and incubating at 37 ℃ for 30 min;
rinsing with PBS for 5min × 3 times, dripping DAB as a color developing agent, rinsing with PBS for 3min × 3 times, counterstaining with hematoxylin for 1min, rinsing with PBS, respectively rinsing with 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, xylene I and xylene II for 1min, sealing with neutral gum, and air drying;
taking a picture by a fluorescence inverted microscope to obtain an immunohistochemical result.
6. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: a25. mu.l reaction system was set up according to the following operating table for QRT-PCR experiments:
。
7. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: detecting dopamine content, SOD activity and MDA content by using striatum tissue suspension, taking 40 mg of cold-stored striatum tissue, homogenizing by a tissue homogenizer, cracking the tissue, preparing 10% homogenate according to the proportion of 1g to 9ml of normal saline after full cracking, centrifuging for 20min at 3000-4000 rmp/min, sucking supernatant, determining protein concentration by a Coomassie brilliant blue method, and subpackaging for later use.
8. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: the method for taking the whole brain tissue comprises the following steps: performing intraperitoneal injection on a rat by using 2% and 0.15 mg/kg of pentobarbital, fully anaesthetizing, fixing the rat on an anatomical plate, sequentially opening an abdominal cavity, fastening all organs with liquid circulation effects below the mediastinum and the chest by using hemostatic forceps, opening the chest, completely exposing the heart, inserting an aortic arch from the left ventricle by using a 7# perfusion needle, rapidly cutting off the right auricle, perfusing 150ml of physiological saline, allowing the blood to become clear, whitening the liver, perfusing and fixing by using PH7.4 and 4% paraformaldehyde by about 100ml, dissecting the brain in time after fixing is completed, taking out brain tissues, fixing in the paraformaldehyde of 4% and PH7.4, and standing by.
9. The method for evaluating the treatment effect of the mesenchymal stem cells on the Parkinson's disease according to claim 1, wherein the method comprises the following steps: the method for directly taking the fresh brain tissue comprises the following steps: intraperitoneal injection is carried out on 2% and 0.15 mg/kg of pentobarbital, after full anesthesia, the neck is quickly cut off, the brain is taken out, rat midbrain and striatum tissues are separated on ice, weighed and frozen at-80 ℃ for later use.
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| CN109954002A (en) * | 2019-03-11 | 2019-07-02 | 北京诺兰药谷科技有限公司 | Application of the human umbilical cord mesenchymal stem cells in preparation prevention Parkinson's disease or the drug for treating Early Parkinson's disease |
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| CN109954002A (en) * | 2019-03-11 | 2019-07-02 | 北京诺兰药谷科技有限公司 | Application of the human umbilical cord mesenchymal stem cells in preparation prevention Parkinson's disease or the drug for treating Early Parkinson's disease |
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