CN114081900A

CN114081900A - Application of nano microvesicle derived from mesenchymal stem cells in preparation of medicine for treating preeclampsia

Info

Publication number: CN114081900A
Application number: CN202110207297.9A
Authority: CN
Inventors: 王凤鸽; 张斌; 满冬梅; 赵柔; 刘淼; 魏利; 梁敬; 陈彦戎; 魏东
Original assignee: AFFILIATED HOSPITAL OF JINING MEDICAL UNIVERSITY
Current assignee: AFFILIATED HOSPITAL OF JINING MEDICAL UNIVERSITY
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2022-02-25

Abstract

The invention provides application of a nano microvesicle derived from mesenchymal stem cells in preparing a medicament for treating preeclampsia, belonging to the technical field of biological medicaments. The invention provides application of a nano microvesicle derived from mesenchymal stem cells in preparing a medicament for treating preeclampsia. Experiments prove that the microvesicles secreted by the umbilical cord mesenchymal stem cells are used for treating a rat animal model at the preeclampsia, and the microvesicles derived from hucMSC have the effects of reducing the blood pressure and 24h proteinuria of a model rat at the preeclampsia, improving the preeclampsia-like symptoms of the rat, increasing the weight of a baby rat, improving the growth of the fetus and providing a new direction for treating preeclampsia diseases.

Description

Application of nano microvesicle derived from mesenchymal stem cells in preparation of medicine for treating preeclampsia

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a nano micro-vesicle derived from mesenchymal stem cells in preparation of a medicine for treating preeclampsia.

Background

Preeclampsia is a serious complication of pregnancy with a morbidity rate of about 5% to 8% and is a major cause of maternal mortality worldwide. Preeclampsia can lead to restricted growth and even death of the fetus. Although preeclampsia has a significant impact on the health of pregnant women and fetuses, there is currently no effective treatment that can extend pregnancy without adverse consequences to the fetus. At present, termination of pregnancy remains the only treatment for preeclampsia, resulting in poor outcome of premature fetal delivery and immature heart and lung. In addition, magnesium sulfate and the like are the most commonly used therapeutic drugs in clinic, but a certain side effect can be caused by long-term large-scale application, and the antihypertensive effect is mainly achieved only from the manifestation symptoms of diseases, but the therapeutic effect is not achieved from the etiology. Therefore, a new preeclampsia treatment scheme is actively explored, the action mechanism of the preeclampsia treatment scheme is disclosed, and the preeclampsia treatment method has important scientific significance and clinical transformation value.

In recent years, Mesenchymal Stem Cells (MSCs) have attracted much attention in the medical field due to their pluripotency and paracrine capacity. However, the ethical, safety, difficulty in storage and transportation, and the like of stem cells limit the clinical application of the stem cells. In recent years, microvesicles, which are important components in paracrine secretion of MSCs, have been reported to be widely involved in many disease physiological and pathological processes such as cell invasion and migration, immunoregulation, angiogenesis, and tissue regeneration. Compared with MSCs, the microvesicles derived from the mesenchymal stem cells have the advantages of convenient transportation, good biological safety, high targeting property and the like. The umbilical cord has also been proved to be one of the high-quality sources of microvesicles secreted by the MSCs, and compared with microvesicles secreted by MSCs from other sources, the microvesicles secreted by the MSC of the umbilical cord have the advantages of no ethical dispute, stable and safe gene, convenient collection, low immunogenicity and the like. The structure of the microvesicles is an important pathway of intercellular signal communication discovered in recent years and is an important paracrine form outside the soluble cell factors of cells. At present, microvesicles derived from MSCs play a role in the repair and regeneration of tissue injury, mainly transmit substances containing proteins, lipids, mRNA, miRNA and the like derived from MSCs and mediate paracrine pathways, so that cells which survive in the injured tissue reenter a cell cycle, and meanwhile, tissue specificity can be obtained through bidirectional exchange with genetic substances of MVs released by the injured cells, and the injured tissue is further repaired. However, there is no report in the prior art that MSCs-derived microvesicles can be used to treat prodigal epilepsy.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of a mesenchymal stem cell-derived nanocapsule in the preparation of a drug for treating preeclampsia.

The invention provides application of a nano microvesicle derived from mesenchymal stem cells in preparing a medicament for treating preeclampsia.

Preferably, the preparation method of the mesenchymal stem cell-derived nano-microvesicle comprises the following steps:

1) performing in-vitro amplification culture on hucMSC, collecting culture supernatant when the cell confluence reaches 75-80%, and removing cell fragments to obtain MSC-CM;

2) removing cell fragments and organelles from the MSC-CM, performing ultrafiltration washing, and collecting a concentrated solution;

3) transferring the concentrated solution to a 30% sucrose/D2O density pad for density gradient centrifugation, and collecting a bottom buffer pad;

4) and diluting and washing the bottom buffer pad, performing ultrafiltration washing, collecting the micro-vesicle concentrated solution, and sterilizing to obtain the nano-micro-vesicle derived from the mesenchymal stem cells.

Preferably, the method of removing cell debris in step 1) comprises centrifugation; the centrifugal force of the centrifugation is 1800-2200 g, and the centrifugation time is 8-12 min.

Preferably, the removal of cell debris and organelles in step 2) comprises differential centrifugation; the centrifugal force of the differential centrifugation is 1500-2200 rpm, and the time of the differential centrifugation is 15-30 min.

Preferably, the ultrafiltration washing in step 2) or step 4) is performed using a 100000Da MWCO ultrafiltration centrifuge tube.

Preferably, the centrifugal force of the density gradient centrifugation in the step 3) is 100000g, and the time of the density gradient centrifugation is 3 h; the temperature of the density gradient centrifugation was 4 ℃.

Preferably, the surface marker proteins of the mesenchymal stem cell-derived nanovesicles include CD63 and CD 9.

Preferably, the medicament is in the form of injection; in the injection, the concentration of the nano-micro vesicle derived from the mesenchymal stem cell is not less than 4.5 multiplied by 10¹¹The number of microparticles was 0.4 ml.

The invention provides application of a nano microvesicle derived from mesenchymal stem cells in preparing a medicament for treating preeclampsia. The secreted microvesicle is extracted from umbilical cord mesenchymal stem cells and used for treating a rat animal model in preeclampsia, and the hucMSC is found to have the effects of reducing the blood pressure and 24h proteinuria of a model rat in preeclampsia, improving the preeclampsia-like symptoms of the rat, and increasing the weight of a baby rat and improving the development of a fetus.

Drawings

FIG. 1 shows the results of the identification of hucMSC microvesicles isolated and purified according to the present invention; wherein FIG. 1A is an electron microscope picture; figure 1B is the results of the size distribution of hucMSC microvesicles; FIG. 1C shows the results of western blot detection;

FIG. 2 is a flow chart of PE rat model modeling and hucMSC microvesicle intervention;

FIG. 3 is a graph showing the result of determining pregnancy in a rat, wherein FIG. 3A is an electron micrograph of a pessary; FIG. 3B is a field view of vaginal secretion; FIG. 3C is a vaginal smear result;

FIG. 4 shows the results of testing rats with a successfully constructed PE model; wherein FIG. 4A is a bar graph of 24h proteinuria levels in rats; FIG. 4B is a histogram of blood pressure in rats;

figure 5 is an improvement in PE rat preeclampsia with hucMSC microvesicle treatment;

figure 6 is a graph of the effect of treatment of preeclampsia with hucMSC microvesicles on PE litter weight, litter size and placental weight, where figure 6A is the average fetal mouse weight and figure 6B is the average placental weight.

Detailed Description

In the present invention, the preparation method of the mesenchymal stem cell-derived nanomicrovesicle preferably comprises the following steps:

2) removing cell fragments and organelles from MSC-CM, then carrying out ultrafiltration washing, and collecting concentrated solution;

3) transferring the concentrated solution to sucrose/D with a mass concentration of 30%₂Performing density gradient centrifugation on the O density cushion, and collecting a bottom cushion;

4) and diluting and washing the bottom buffer pad, performing ultrafiltration washing, collecting concentrated solution, and sterilizing to obtain the nano microvesicle from the mesenchymal stem cells.

The invention carries out in-vitro amplification culture on hucMSC, collects culture supernatant when the cell confluence reaches 75-80%, and removes cell fragments to obtain MSC-CM.

In the present invention, the method for the in vitro amplification culture of hucMSCs can be found in the prior art (Qiao Chun et al, human mesenchyme stem cells isolated from the said universal code. cell Biol int.2008 Jan; 32(1): 8-15.). In the present examples, hucMSCs were purchased from race organisms. The hucMSC is preferably a healthy human hucMSC having a high proliferation ability, for example, a third-generation or fourth-generation cell selected for in vitro amplification culture. The culture preferably adopts a serum-free culture medium.

In the present invention, the method of removing cell debris preferably comprises centrifugation; the centrifugal force of the centrifugation is preferably 1800-2200 g, and more preferably 2000 g; the time for centrifugation is preferably 8-12 min, and more preferably 10 min.

After obtaining the MSC-CM, the invention removes cell fragments and organelles from the MSC-CM, then carries out ultrafiltration washing, and collects concentrated solution.

In the present invention, removing cell debris and organelles preferably comprises differential centrifugation; the centrifugal force of the differential centrifugation is preferably 1500-2200 rpm, and more preferably 2000 rpm. The time of the differential centrifugation is preferably 15-20 min, and more preferably 20 min. The ultrafiltration washing is preferably carried out using a 100000Da MWCO ultrafiltration centrifuge tube. The source of the 100000Da MWCO ultrafiltration centrifugal tube is not particularly limited, and the source of the 100000Da MWCO ultrafiltration centrifugal tube known in the field can be adopted.

After the concentrated solution is obtained, the invention transfers the concentrated solution to sucrose/D with the mass concentration of 30 percent₂Density gradient centrifugation was performed on the O density pad and the bottom cushion was collected.

In the present invention, the centrifugal force of the density gradient centrifugation is preferably 100000g, and the time of the density gradient centrifugation is preferably 3 h; the temperature of the density gradient centrifugation is preferably 4 ℃. The invention adds 30% sucrose/D₂The O density pad is not particularly limited and is prepared using 30% sucrose/D by mass as is well known in the art₂And preparing the O density pad. Microvesicles were settled to the bottom by density gradient centrifugation, collecting the bottom buffer for subsequent purification.

After the bottom buffer pad is obtained, diluting and washing the bottom buffer pad, performing ultrafiltration washing, collecting a micro-vesicle concentrated solution for sterilization to obtain the nano micro-vesicle from the mesenchymal stem cells.

In the present invention, the diluted washing solution is preferably 7.4 value PBS buffer. The bottom buffer pad is preferably diluted by 2-10 times, more preferably 10 times, with PBS buffer.

In the present invention, the ultrafiltration washing is preferably to transfer the obtained dilution to a 100000Da MWCO ultrafiltration centrifuge tube and collect the microvesicle concentrate. The sterilization is preferably performed by filtering with a filter membrane; the pore size of the filter is preferably 0.22 μm.

In the present invention, it is preferable to identify the obtained mesenchymal stem cell-derived nanomicrovesicles. The identification includes basic morphology identification and identification of surface marker proteins. The basic morphological identification comprises the observation of separated and purified nano-micro vesicles by a transmission electron microscope. The transmission electron microscope picture shows that the microvesicle has a typical vesicle structure, and the particle size is about 100 nm. The identification of the surface marker protein is preferably detected by adopting a western blot technology. The protein detected by the western blot technique preferably comprises CD63 and CD9 and a negative marker Calnexin. The result shows that the surface marker protein of the mesenchymal stem cell-derived nano-microvesicle obtained by separation and purification of the invention preferably comprises CD63, CD9 and Calnexin.

In the present invention, the dosage form of the drug is preferably an injection; in the injection, the concentration of the mesenchymal stem cell-derived nanovesicles is preferably not less than 4.5 × 10¹¹The number of microparticles was 0.4 ml.

In the invention, a rat model in preeclampsia is constructed, the nano-microvesicles derived from mesenchymal stem cells obtained by separation and purification are respectively treated with low dose and high dose, and the results of measurement of the urine protein level and the blood pressure of a rat show that the high dose of hucMSC microvesicles can obviously reduce the blood pressure of the rat and the 24h protein urine level, while the low dose of hucMSC microvesicles can obviously reduce the blood pressure of the rat, but the statistical difference of the 24h urine protein level is not obvious and only shows a descending trend. Meanwhile, the invention also evaluates the influence of the treatment of the hucMSC microvesicles on the weight of the fetus and the placenta, and the results show that the average weight of the newborn mouse can be increased by the high-low dose treatment group, and the average placenta weight can be increased by the high-dose treatment group, so that the development outcome of the fetus can be improved.

The application of the mesenchymal stem cell-derived nanocapsule in the preparation of a medicament for treating preeclampsia provided by the present invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Method for obtaining hucMSC Microvesicles (MVC)

1. The main materials and sources are respectively as follows:

1.1 reagent: low-sugar DMEM, trypsin (product of Sigma), fetal bovine serum (product of Gibco), serum-free medium (shanghai enciose), microvesicle extraction reagent: deuterium oxide (D2O, shanghai seiko), analytically pure sucrose (guangzhou chemical reagent factory), rabbit anti-human CD9 antibody (Bioworld Technology, usa), rabbit anti-human CD63 antibody (Epitomics, usa), rabbit anti-human Calnexin antibody (abcam, usa), BCA protein quantification kit, horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG secondary antibody (beijing kang, century), HRP chemiluminescent substrate;

1.2 instruments, equipment and consumables: carbon dioxide incubator (Forma corporation); a bench top gradient centrifuge, an electron microscope, an inverted microscope, a biomicroscope, an ultraclean bench top, a 100-kDa MWCO ultrafiltration centrifuge tube, a 0.22 μm sterile filter membrane (Millipore, USA), a transmission electron microscope (FEI Tecnai 12, Philips), a filter membrane.

2. Experimental methods

(1) Isolated culture of hucMSC:

in vitro culture and amplification of MSC (Qiao Chun et al, human sensory cellular cells isolated from the national cord. cell Biol int.2008 Jan; 32(1):8-15) according to the hucMSC isolation culture and identification method established earlier in this subject group, selecting healthy human hucMSC (third generation or fourth generation) with strong proliferation ability, removing serum-free culture medium after the growth area covers 80%, collecting culture supernatant, centrifuging at 2000g/10min to remove cell fragments to obtain huSC-CM, and storing at 80 ℃ for extraction of microvesicles.

(2) Separating and purifying umbilical cord mesenchymal stem cell microvesicles:

differential separating the collected hucMSC-CM, removing cell debris and organelles, transferring to 100000Da MWCO ultrafiltration centrifugal tube with 15mL specification, concentrating, transferring the concentrated solution to 5mL sucrose/D with concentration of 30%₂Centrifuging at 100000g for 3h on O density pad at 4 deg.C, collecting buffer pad (containing microvesicle) with bottom 5ml, diluting and washing with PBS, washing in 100000Da MWCO ultrafiltration centrifuge tube, and quantifying the collected microvesicle concentrateFiltering with 0.22 μm filter membrane for sterilization, measuring the number of micro-particles of the cell microvesicle by Nansigt method, packaging, and refrigerating at-80 deg.C.

(3) Identification of umbilical cord mesenchymal stem cell microvesicles

And (3) observing the basic morphology of the umbilical cord mesenchymal stem cell microvesicle obtained by separation and purification by using a transmission electron microscope, wherein the specific method comprises the following steps: 20 mu L of umbilical cord mesenchymal stem cell microvesicle is fully and uniformly mixed, then the microvesicle is dripped on a sample-carrying copper net with the diameter of 2mm, after the microvesicle is kept still for 5min at room temperature, the residual liquid at the edge of the copper net is slightly absorbed by using filter paper, then the copper net is reversely covered on 30g/L phosphotungstic acid (pH 6.8) liquid drops, the microvesicle is negatively dyed for 5min at room temperature, finally the copper net is dried under an incandescent lamp, and the microvesicle is placed under a transmission electron microscope for observation and photographing, and the result is shown in the attached figure 1-A.

The method for detecting the micro vesicle surface marker protein by adopting a western blot technology comprises the following steps: preparing 12% SDS-PAGE electrophoresis gel, fully cracking the extracted microvesicles, adding 1/4 volumes of 5 xSDS loading buffer solution, boiling for 5min, loading according to 200 mug protein total amount, transferring the proteins onto a PVDF membrane by electrotransfer (350 mA, 120min), sealing for 1h at room temperature with TBS/T containing 50g/L skimmed milk, reacting with rabbit anti-human CD9 antibody and rabbit anti-human CD63 antibody (1:500) at 4 ℃ overnight, washing the membrane for 3 times with TBS/0.5% Tween 20 for the next day, incubating with HRP-labeled goat anti-rabbit IgG secondary antibody at 37 ℃ for 1h, washing the membrane for 3 times with TBS/0.5% Tween 20, adding HRP premixed chemiluminescent substrate, and detecting by a chemiluminescent gel imaging system, wherein the expression of the labeled proteins of the microvesicles derived from umbilical cords is shown in figure 1C.

Example 2

Method for constructing preeclampsia rat model

1. Materials and reagents: healthy SD female rats (200-250 g) and male rats (200-250 g) at the age of 10 weeks are purchased from Jinan Pengye laboratory animals Co., Ltd, common feed (Jinan Pengye laboratory animals Co., Ltd), disposable syringes, L-NAME, physiological saline, rat metabolic cages, fully automatic biochemical analyzers (Roche, Switzerland), fully automatic noninvasive blood pressure monitors (Softron, Beijing, China), and the like.

2. Experimental method (see step fig. 2):

construction of rat eclampsia model: healthy SD female rats and male rats aged 10 weeks are fed for 1 week, numbered after adapting to the environment, and are subjected to cage combination at 8h in a night according to a male-female ratio of about 2: 1. The following morning, 7 points after separation of the males and females, vaginal suppositories were examined. If the pessary is seen (FIG. 3A) it is considered the first day of pregnancy. Vaginal secretions (fig. 3B) were removed without visualization of the pessary and vaginal smears were made to observe sperm and epithelial cells (fig. 3C). If sperm are present on the slide, the day is considered to be the first day of pregnancy.

Pregnant rats were randomly divided into 2 groups: normal Control group (Control), PE model group; the mice in the PE model group, the high-concentration microvesicle group and the low-concentration microvesicle group were injected with L-NAME (nitroso-L-arginine methyl ester) at 250mg/kg/d intraperitoneally from day 7 to 19 of pregnancy. Normal control groups were injected intraperitoneally with normal saline from day 7 to day 19 of pregnancy. And blood pressure on 6 th (day before L-NAME injection), 10 th and 13 th day of pregnancy was monitored by BP2010 full-automatic noninvasive blood pressure monitor. On day 6 of pregnancy (the day before injection of L-NAME), day 10 and day 13, 24-hour urinary protein levels were measured in rats using a fully automatic biochemical analyzer.

The results showed that the blood pressure in the model group was elevated more than 30mmHg from the normal group (fig. 4A), and the urinary protein level was significantly elevated at 24 hours (fig. 4B). The results show that the PE model rat is successfully constructed.

Example 3

Evaluation of therapeutic Effect of hucMSC microvesicles on Pre-eclamptic rats

1. Required materials and reagents: the kit comprises a disposable syringe, hucMSC microvesicles, physiological saline, a rat metabolism cage, a full-automatic biochemical analyzer, a full-automatic noninvasive blood pressure monitor, pentobarbital sodium and a dosage balance.

2. Experimental methods

Treatment procedure and efficacy evaluation of HucMSC microvesicles:

four groups are set:

control group: 14 tail vein injection of physiological saline is started, 0.4 ml/rat/day, and 6 days are total;

PE group: starting to inject normal saline into tail vein at 14 th day, wherein the normal saline is 0.4 ml/rat/day, and the total amount of the normal saline is 6 days;

low concentration microvesicle set (PE + L-MVS): the amount of each tail vein injection (0.4ml) was 2.25X 10 from 14 th¹¹Microvesicle suspension with particle number, 0.4 ml/rat/day, total 6 days;

high concentration microvesicle set (PE + H-MVS): the amount of each tail vein injection (0.4ml) was 4.5X 10 from 14 th¹¹Microvesicles suspension with particle number, 0.4 ml/rat/day, for 6 days.

1) Blood pressure and 24 hour urinary protein levels were monitored on days 16 and 19 of pregnancy, respectively.

The results are shown in Table 1 and FIG. 5.

TABLE 1 blood pressure and proteinuria test results of rats in each experimental group and control group

Note: all data are expressed as "mean ± standard deviation", with different letters in the same row indicating a significant level of difference of 0.01.

The results show that the high dose of the hucMSC microvesicle can obviously reduce the blood pressure and 24h proteinuria level of the rat, and the low dose of the hucMSC microvesicle can obviously reduce the blood pressure of the rat but has no obvious statistical difference but has a descending trend in the 24h proteinuria level.

2) Treatment of HucMSC microvesicles was assessed on fetal and placental weight with observations of effect:

after anesthetizing the rats, 3% pentobarbital sodium solution is used, and after anesthetizing, cesarean section is immediately performed to take out the fetuses and placentas, sew the uteri and the abdomen, and count the fetuses and placentas with normal sizes. Removing the fetal membrane and umbilical cord from placenta, cutting off umbilical cord, placing the fetus and placenta on sterile gauze, and sucking to remove blood and amniotic fluid. Subsequently, the fetus and placenta were weighed separately on analytical balances.

The results are shown in Table 2 and FIG. 6.

TABLE 2 weight of fetal rat and placenta

	Control	PE	PE+L-MVS	PE+H-MVS
					Fetal rat weight (g)	3.006±0.103^a	2.635±0.245^b	2.932±0.086^a	2.981±0.058^a
Placenta weight (g)	0.453±0.051^a	0.351±0.046^b	0.389±0.038^ab	0.429±0.051^a

Note: all data are expressed as "mean ± standard deviation", with different letters in the same row indicating a significance level of 0.05.

The results show that the average weight of the newborn mice can be increased in the high-low dose treatment group, and the average placenta weight can be increased in the high-dose treatment group, so that the development outcome of the fetus can be improved.

From the results of the above examples, it can be seen that the treatment of preeclampsia diseases by using the HucMSC microvesicle not only can improve the blood pressure and urine protein indexes of pregnant mothers, but also can significantly improve the weight of fetuses and the weight of placentas, and can improve the development conditions of fetuses.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of the nano microvesicle derived from the mesenchymal stem cells in preparing a medicament for treating preeclampsia.

2. The use according to claim 1, wherein the preparation method of mesenchymal stem cell-derived nanomicrovesicles comprises the following steps:

4) and diluting and washing the bottom buffer pad, performing ultrafiltration washing, collecting a micro-vesicle concentrated solution, and sterilizing to obtain the nano micro-vesicle derived from the mesenchymal stem cells.

3. Use according to claim 2, wherein the method of removing cell debris in step 1) comprises centrifugation; the centrifugal force of the centrifugation is 1800-2200 g, and the centrifugation time is 8-12 min.

4. The use according to claim 2, wherein the removal of cellular debris and organelles in step 2) comprises differential centrifugation; the centrifugal force of the differential centrifugation is 1500-2200 rpm, and the time of the differential centrifugation is 15-30 min.

5. The use of claim 2, wherein the ultrafiltration washing in step 2) or step 4) is performed using a 100000Da MWCO ultrafiltration centrifuge tube.

6. The use of claim 2, wherein the centrifugal force of the density gradient centrifugation in step 3) is 100000g, and the time of the density gradient centrifugation is 3 h; the temperature of the density gradient centrifugation was 4 ℃.

7. The use of any one of claims 2 to 6, wherein the surface marker proteins of the mesenchymal stem cell-derived nanovesicles comprise CD63 and CD 9.

8. The use of claim 7, wherein the medicament is in the form of an injection;

in the injection, the concentration of the nano-micro vesicle derived from the mesenchymal stem cell is not less than 4.5 multiplied by 10¹¹The number of microparticles was 0.4 ml.