CN110042127A - Preparation, preparation method and the application of cells into cardiomyocytes delivering target nucleic acid molecules - Google Patents
Preparation, preparation method and the application of cells into cardiomyocytes delivering target nucleic acid molecules Download PDFInfo
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Abstract
The present invention relates to preparation, preparation method and the applications of a kind of cells into cardiomyocytes delivering target nucleic acid molecules.The delivering refers to the cardiac muscle cell's intake for improving drug, enhances heart local drug concentration；The preparation refers to CHO-PGEA- nucleic acid complex preparation.
Description
Technical field
The invention belongs to field of pharmaceutical biology, in particular to a kind of cells into cardiomyocytes delivering target nucleic acid molecules
Preparation, preparation method and application.
Background technique
In the world, cardiovascular disease is still the main reason for leading to death, and illness rate and age are presented just
Correlation increases.With the growth of aging of population, the illness rate of cardiovascular disease be increased significantly.Although with medical level
It is promoted, we have obtained first-stage success to the preventing and controlling of cardiovascular disease.But in general, morbidity and mortality are still
Cumulative year after year.Wherein, chronic heart failure is often hypertension, aortic stenosis, myocardial infarction, a variety of angiocarpy such as cardiomyopathy
The terminal final result of disease.Heart failure is mainly reduced using heart function and heart reconstruction is pathologic, physiologic feature.Existing clinic is controlled
Although treatment method can be relieved the clinical symptoms of patients with heart failure, but cannot prevent or reverses cardiac muscle cell in molecular level change,
And 5 annual death rates of patients with heart failure are still high, not acceptable.
Compared to the treatment of conventional medicament, gene therapy has the targeting for particular biological process, this make it
Treating has breakthrough meaning in heart disease.And gene therapy is in a variety of heart diseases such as heart failure and hypertrophic cardiomyopathy
In application, be but limited to drug to the low transfection efficiency of cardiac muscle cell.
Ethanol amine-amination poly- (glycidyl methacrylate) (CHO-PGEA) of cholesterol (CHO) sealing end is a kind of
The cationic polymer of novel synthesis can be used as the nano-carrier of delivering nucleic acid molecules.Its principle is formed by the way that nucleic acid is complexed
CHO-PGEA/ nucleic acid nano compound.Since the nanoparticle surface is negatively charged, electronegative cell can be electrostatically attracted to
Film shows to help them by cell internalizing.
It has been confirmed currently, CHO-PGEA enters in tumour cell for effectively delivering Plasmid DNA.Compared to it is poly- sun from
The goldstandard of sub- genes delivery system --- polyethyleneimine (PEI), CHO-PGEA have more efficient cell internalizing ability, can
More nucleic acid molecules are carried to enter in tumour cell.But whether CHO-PGEA can be used as nano-carrier, and nucleic acid high-efficiency delivery is arrived
It is not proven in cardiac muscle cell.It is worth noting that, the characteristic structural of cardiac muscle cell is lateral tubule (TT), it is by invaginating
Myocardial cell membrane formed, increase myocardial cell surface product and volume ratio.[50]In addition, cholesterol level in lateral tubule
It is extremely abundant, be conducive to CHO-PGEA/ nucleic acid complexes using its particle surface cholesterol group, by with myocardial cell membrane
Similar intermiscibility enters intracellular.
In view of the above problems, present inventor passes through animal model to CHO-PGEA/ nucleic acid complexes in cardiac muscle cell
In transfection efficiency verified, and confirm that CHO-PGEA/ nucleic acid is multiple using the miR182 inhibitor of targeting myocardial hypertrophy
Object is closed to the therapeutic effect of heart disease.
Summary of the invention
Present invention firstly relates to CHO-PGEA carriers in the preparation for preparing cells into cardiomyocytes delivering target nucleic acid molecules
Using the target nucleic acid molecules include but is not limited to:
(1) artificial synthesized or naturally occurring for adjusting the single-stranded or double-stranded DNA molecular of cardiac function；
(2) artificial synthesized or naturally occurring for adjusting the coding or non-coding RNA molecule of cardiac function；
(3) artificial synthesized or naturally occurring for treating the single-stranded or double-stranded DNA molecular of cardiovascular disease；
(4) artificial synthesized or naturally occurring for treating the coding or non-coding RNA molecule of cardiovascular disease；
(5) the artificial synthesized RNA analogies and antisense oligonucleotides acid fragment for being used to adjust cardiac function.
The delivering refers to the intake for improving cardiac muscle cell to target nucleic acid molecules, and it is especially myocardium to improve heart part
The concentration of intracellular target nucleic acid molecules.
Preferably, the nitrogen of the carrier molecule in the CHO-PGEA carrier/phosphorus ratio is 10.
The invention further relates to a kind of pharmaceutical composition for treating heart disease, the treatment refers in animal or human body
Cardiac function is repaired, heart reconstruction symptom and sign is mitigated or improves the symptom and sign that heart disease causes, the pharmaceutical composition
Object includes the preparation and conventional pharmaceutic adjuvant of therapeutically effective amount.
The invention further relates to application of the preparation in the drug of preparation treatment heart disease, the treatment refers to
Cardiac function is repaired in animal or human body, mitigates heart reconstruction symptom and sign or improves the symptom and sign that heart disease causes.
Detailed description of the invention
Cell internalizing effect of Fig. 1, CHO-PGEA/ nucleic acid complexes in cardiac muscle cell.
1a successfully absorbs cardiac muscle cell (positive cell) number ratio of CHO-PGEA/ nucleic acid complexes.
1b, the concentration of CHO-PGEA/ nucleic acid complexes in positive cell.
1c, the fluorescence distribution figure of different carriers-miRNA compound in cardiac muscle cell.
Distribution map of Fig. 2, CHO-PGEA/ nucleic acid complexes in heart tissue.
2a, by angular vein injecting normal saline, dissociate miR-Cy3, PEI/miR-Cy3 and CHO-PGEA/miR-Cy3
The mouse heart tissue fluorogram of 1h afterwards.
2b, to the statistical chart of relative radiative efficiency in a figure.
2c after heart tissue carries out frozen section, uses the fluorogram of fluorescence microscope shooting.
Therapeutic effect of Fig. 3, CHO-PGEA/ nucleic acid complexes to heart failure mouse.
3a, heart is substantially schemed and cardiac mass and tibia length ratio (HW/TL).
3b, heart tissue sections tritin (WGA) dyeing and cardiomyocyte surface area statistics.
The postoperative 9 weeks mouse M- type echocardiographic images of 3c, TAC.
The postoperative 3-9 weeks mouse ejection fraction (EF) of 3d, TAC, left room short axle shortens score (FS) and left room end-systole is straight
Diameter (LVIDs) statistical value.
The protein expression situation of FOXO3 in the postoperative 9 weeks mouse hearts of 3e, TAC.
Specific embodiment
Experimental animal, reagent and kit, instrument
Experimental animal
No-special pathogen (Specific Pathogen Free, SPF) grade Male wild-type (Wild Type, WT)
C57BL/6J background mice is purchased from Fukang Biotechnology Co., Ltd, Beijing China (BeiJing, China).All mouse raisings are in Beijing
In SPF grade environment under cardiopulmonary and vascular disease research institute animal house controlled temperature, and it can freely obtain food and water.All experiments
Animal uses 10-12 week old male WT mouse, and body weight control is between 20-25g.Animal protocols are by the Capital University of Medical Sciences
Animal care and ratified using the committee, and meets National Institutes of Health (National Institutes of
Health, NIH) " management of laboratory animal and guide for use " (NIH publication the 85-23rd, 2011 revise) formulated.Although
Priori efficiency analysis is not carried out, but in order to abide by animal welfare regulation, has used the animal of limited quantity.Empirical numerical is based on
The experience of previously used animal model.In treatment end, by amobarbital (200mg/kg, intraperitoneal injection) to mouse
Implement euthanasia.
Biochemical reagents and kit title and supplier see the table below 1
Table 1, biochemical reagents and kit title and supplier
Experimental instrument and equipment title and supplier see the table below 2
Table 2, experimental instrument and equipment title and supplier
The cell internalizing effect of embodiment 1, CHO-PGEA/ nucleic acid complexes in cardiac muscle cell.
Detect cell internalizing effect of the CHO-PGEA/ nucleic acid complexes in cardiac muscle cell.
Cell internalizing effect is evaluated in High content screening system statistical analysis.
Cultivate embryo's rat myocardial cell system (H9C2 cell line) in 24 orifice plates, every hole be added 20 μ L containing polycation/
MiR-Cy3 compound.Wherein the preparation method of CHO-PGEA/miR-Cy3 compound and PEI/miR-Cy3 compound referring to
CN201510377896.X.It is prepared with 5 to 20 N/P ratio (N/P).The mixing of compound according to the form below ratio, oscillation are stood after 5 seconds
30 minutes:
Replacement fresh culture continues culture 20 hours after culture 4 hours.The cell cultivated is washed with PBS twice, and
It is incubated at room temperature cell 10 minutes using DAPI (being diluted to 150ng/ml concentration 1 using PBS), nucleus is dyed.It uses
MataXpress software is made carefully with High Content Scanner (Molecular Devices, Silicon Valley, CA)
Born of the same parents visualize and quantify.
The results show that
(1) compared with free miR-Cy3, N/P ratio is 10 PEI/miR-Cy3 compound, CHO- of all N/P than under
PGEA/miR-Cy3 compound shows better cellular uptake ability (Fig. 1 a).
(2) when N/P ratio is 5, in the H9C2 with the processing of CHO-PGEA/miR-Cy3 compound, Cy3 fluorescence is given out
Positive cell percentage is more than 67%.
(3) when N/P ratio is 10,15,20, for the percentage of positive cell more than 86%, this ratio is much higher than PEI/
MiRNA compound treated cell, also, after N/P is greater than 10, the increase of N/P ratio can not lead to cellular uptake miRNA
Amount it is linearly increasing.
(4) and when N/P is 10, after the processing of CHO-PGEA/miR-Cy3 compound, the average fluorescent strength ratio of cell
Strong 1.5 times (Fig. 1 b) of cell handled using PEI/miR-Cy3 compound.
In addition, compound to polycation/miR-Cy3 using confocal laser scanning microscope, CLSM (CLSM) assessment H9C2 cell
The intake ability of object.Free miR-Cy3, the PEI/miR-Cy3 and CHO- that N/P is 10 are separately added into H9C2 cell
PGEA/miR-Cy3.After 24 hours, by DAPI dyestuff by nucleus au bleu, and the miR-Cy3 for entering cell then may be used
Red fluorescence is inspired by fluorescence microscope.It is observed by CLSM, it has been found that multiple with free miR-Cy3 and PEI/miR-Cy3
It closes object to compare, more CHO-PGEA/miR-Cy3 compounds can be observed in H9C2 cell, this proves that CHO-PGEA has more
High cellular uptake ability (Fig. 1 c).
According to above-mentioned data, it is known that miRNA can be effectively transported in cardiac muscle cell by CHO-PGEA.In addition, we
Early-stage study show CHO-PGEA have cell internalizing ability more better than the other polymers of not cholesterol modification, say
The clear cholesterol group importance in gene delivery in the cell.
The heart distribution of embodiment 2, CHO-PGEA/ nucleic acid complexes.
The male WT mouse of C57BL/6J background is bought from Fukang Biotechnology Co., Ltd, Beijing China, and is maintained at disease-free
It is fed in the controlled heat environment of substance.All test carries out in the male WT mouse using 10-12 week old, and animal protocols are by head
All medical university's animal care and the approval using the committee.
12 C57BL/6J mouse are randomly divided into 4 groups in total, are respectively as follows:
1. blank control group: every mouse injects 100 μ L physiological saline.
2. negative control group: dissolving 5nmol microON using 100 μ L DEPC waterTM mimic Negative
Control#22 (5Cy3) i.e. miR-Cy3 freeze-dried powder.The 5nmol miR-Cy3 of every mouse injection dissolution.
3. experimental group: injection 100 μ L nitrogen/phosphorus (contains 5nmolmiR- than the CHO-PGEA/miR-Cy3 compound for 10
Cy3)。
4. positive controls: injection 100 μ L nitrogen/phosphorus is than the PEI/miR-Cy3 compound (containing 5nmolmiR-Cy3) for 10.
Configured each group reagent treatment is passed through angular vein respectively to be injected into Mice Body.After 1 hour, mouse core is taken
It is dirty, it is glimmering that polycation/miR-Cy3 compound in heart is captured by Xenogen IVIS small animal living body optical imaging system
Optical signal, and analyzed.The results show that after injection CHO-PGEA/miR-Cy3, the significant increase of the fluorescence intensity of heart tissue,
And it is better than negative and positive controls mouse heart fluorescence intensity (Fig. 2 a, b).As shown in Figure 2 b, miR-Cy3 transmitting is collected
Heart fluorescence, and quantitative analysis is carried out by relative radiative efficiency.Compared with PEI/miRNA-Cy3 compound processing group, CHO-
The radiation efficiency of PGEA/miRNA-Cy3 compound processing group improves 2 times or more.
After completing optical imagery, frozen section is made in mouse heart tissue as early as possible, and adopt figure under fluorescence microscope.Knot
Fruit shows that the mouse heart for giving miR-Cy3 processing can hardly observe red fluorescence.PEI/miR-Cy3 processing group mouse
In heart sections, although it can be observed that the red fluorescence that miR-Cy3 is issued, it accumulates degree and is significantly lower than CHO-PGEA/
The mouse heart tissue (Fig. 2 c) of miR-Cy3 compound processing.
These statistics indicate that, CHO-PGEA can protect miRNA from the degradation of RNase in blood, and can be by miRNA
Successful delivery is into heart tissue.And the delivery efficiency of internal CHO-PGEA is substantially better than the delivery efficiency of PEI.
Embodiment 3, CHO-PGEA/ nucleic acid complexes are to the curative effect of heart disease
Detect the curative effect of CHO-PGEA/ nucleic acid complexes.
15 wild mouses are selected to be divided into two big groups, comprising:
(1) 5 mouse of blank control group: the injecting normal saline in sham-operation (sham) group mouse；
(2) 5 mouse of negative control group: in 3 weeks Mice Bodies of TAC model, injecting N/P ratio by angular vein is
10 CHO-PGEA/NCinhibitor (CHO-PGEA/NC-in) compound, each 100 μ L of dosage (contain 5nmol's
NCinhibitor), every 3 days it is primary, continuous injection 6 weeks.
(3) after TAC model 3 weeks in Mice Body, the CHO- that N/P ratio is 10 experimental mice: is injected by angular vein
PGEA/miR-182inhibitor (CHO-PGEA/miR-182-in) compound, each 100 μ L of dosage (contain 5nmol's
MiR-182inhibitor), every 3 days it is primary, continuous injection 6 weeks.
Observation index includes: after administration
1. cardiac function changes: testing ultrasonic cardiography graph evaluation mouse heart function once every 2 weeks upon administration.
Observe terminal:
At modeling 9 weeks, echocardiogram is being left and taken, after weighing in, is putting to death each group mouse.It collects heart tissue and weighs
Quality.
Transthoracic echocardiography detection: before transthoracic echocardiography, by the chest shaving of mouse and depilatory cream is used
(Veet, Reckitt Benckiser, China) processing.With the ethobrom anesthetized mice of saturation.In order to measure global heart
Function and left ventricular mass carry out echocardiography to mouse in 3,5,7 and 9 weeks before TAC and after TAC.Use Visual
2100 high-resolution ultrasound imaging system of Sonic acquires M-mode image.
Substantially data statistics: it at the end of experiment, is anaesthetized using yellow Jackets (100mg/kg) and puts to death mouse.Physiology salt
After water perfused hearts, heart weighing is taken out, shin bone is taken out and measures length.Data are recorded, and calculate cardiac mass/tibia length ratio
It is worth and counts.
Histopathology dyeing:
I tritin (WGA) dyeing observation cardiac muscle cell's plumpness degree:
(1) slice dewaxing is to water: slice is completely soaked in xylene solution, is taken out after twenty minutes, then be soaked in new two
In toluene solution, it is repeated 2 times completion dewaxing.Slice is taken out from dimethylbenzene and is soaked in 100% dehydrated alcohol, is taken out after five minutes.
It is soaked in 95% ethanol solution again, takes out after five minutes.It is soaked in 80% ethanol solution again, takes out within 5 minutes, completes dehydration.
(2) ethyl alcohol adhered on slice wash with distilled water.
(3) antigen retrieval: using heating in 1 × citric acid solution (about 600mL) pressure cooker, timing 1 divides 30 seconds after boiling,
Guan Huozhi natural cooling.
(4) three times using 1 × PBS cleaning.
(5) slice is prevented in the wet box being protected from light, is closed 30 minutes using 3%BSA room temperature.
(6) WGA is taken out from -20 DEG C of refrigerators, is diluted using 1 × PBS by 1:200.Extra BSA is softly wiped, drop dye is dilute
WGA after releasing is protected from light and is incubated for 1 hour at 37 DEG C.
(7) DAPI mounting fluid-tight piece is used.It is protected from light lower use Nikon ECLIPSE 90i fluorescence microscope and adopts figure.
Cardiomyocyte surface area is detected using NIS-ELEMENTS quantitative analysis software.
II haematoxylin eosin stains observe other organ-tissue morphological changes, assess safety:
By CHO-PGEA/miRNA treated TAC model mice, collects liver, kidney, lung, spleen and paraffin is respectively prepared and cuts
Piece.
(1) by heart paraffin section de-waxing to water
(2) hematoxylin dyes 3 minutes；Flowing water rinses 1 minute.
(3) 1% acidic alcohols break up liquid and break up 1-3 seconds, are put into rapidly in flowing water and rinse 5 minutes anti-indigo plant.
(4) 0.5% Yihong liquid dye 1-2 minutes, and flowing water rinses 30 seconds.
(5) 80% ethyl alcohol impregnate 30 seconds, and 95% ethyl alcohol impregnates 30 seconds, and 100% ethyl alcohol impregnates 5 minutes.
(6) xylene soak 10 minutes.
(7) neutral gum sealing.
(8) after drying in draught cupboard, figure is adopted under the microscope.
Western blot analysis:
It is prepared with the lysis buffer containing complete protease inhibitor mixture (Roche, Mannheim, Germany)
The histone lysate of left ventricle sample measures protein content, and albumen is denatured by boiling (99 DEG C, 5 minutes).Pass through SDS-
Simultaneously electricity is gone on nitrocellulose filter PAGE electrophoretic separation, is used 5% milk (2.5g skimmed milk power is dissolved in 50mlTBST solution)
Closing 30 minutes, then using primary antibody (anti-FOXO3 (1:500) and anti-GAPDH (1:1000)) 4 DEG C of overnight incubations.Second day room
After temperature balance 30min, washed 3 times using 1 × TBST, secondary antibody (1:10000, the Rockland being conjugated with IR dyes
Immunochemicals, Gilbertsville, PA), it is incubated at room temperature 1 hour.After 1 × TBST is cleaned 3 times, use
Odyssey infrared imaging system (LI-COR Biosciences Lincoln, NE) is quantitative.
The results show that CHO-PGEA/miR-182-in processing group mouse reduces compared with the mouse heart relative mass of control group
(Fig. 3 a), WGA coloration result show that plump degree is substantially reduced (Fig. 3 b).Ultrasonic experiments prompt experimental mice cardiac ejection point
Number (EF) and shortening score (FS) maintain to stablize, and negative control group then extends lasting reduction with TAC model time, illustrates reality
Test group mouse heart function be improved significantly (Fig. 3 c, d).Protein immunoblotting is the results show that give CHO-PGEA/miR-
After 182-in, the protein expression of the target gene FOXO3 of miR-182 is gone up (Fig. 3 e), illustrates that the compound successfully inhibits
The function of miR-182 improves molecular pathway variation of the cardiac muscle cell under plump stimulation.
Finally, it should be noted that above embodiments are used only as helping skilled in the art to understand technical solution of the present invention
Essence, limiting the scope of the present invention that it goes without doing.
Claims (6)
- Application of the 1.CHO-PGEA carrier in the preparation for preparing cells into cardiomyocytes delivering target nucleic acid molecules.
- 2. application according to claim 1, which is characterized in that the target nucleic acid molecules include:(1) artificial synthesized or naturally occurring for adjusting the single-stranded or double-stranded DNA molecular of cardiac function；(2) artificial synthesized or naturally occurring for adjusting the coding or non-coding RNA molecule of cardiac function；(3) artificial synthesized or naturally occurring for treating the single-stranded or double-stranded DNA molecular of cardiovascular disease；(4) artificial synthesized or naturally occurring for treating the coding or non-coding RNA molecule of cardiovascular disease；(5) the artificial synthesized RNA analogies and antisense oligonucleotides acid fragment for being used to adjust cardiac function.
- 3. application according to claim 1 to 2, which is characterized in that the delivering, which refers to, improves cardiac muscle cell to mesh The intake of nucleic acid molecules is marked, the concentration of the target nucleic acid molecules in the especially cardiac muscle cell of heart part is improved.
- 4. application according to claim 1 to 2, which is characterized in that the carrier molecule in the CHO-PGEA carrier Nitrogen/phosphorus ratio be 10.
- 5. a kind of pharmaceutical composition for treating heart disease, the treatment refer to repaired in animal or human body cardiac function, Mitigate heart reconstruction symptom and sign or improve the symptom and sign that heart disease causes, the pharmaceutical composition includes that treatment is effective Any preparation of the claim 1-4 of amount and conventional pharmaceutic adjuvant.
- 6. application of any preparation of claim 1-4 in the drug of preparation treatment heart disease, the treatment refer to Cardiac function is repaired in animal or human body, mitigates heart reconstruction symptom and sign or improves the symptom and sign that heart disease causes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111184734A (en) * | 2020-03-05 | 2020-05-22 | 北京市心肺血管疾病研究所 | Application of miRNA in treatment of myocardial hypertrophy |
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Application publication date: 20190723 |