CN111150856A - Ultrasonic molecular probe and preparation method thereof - Google Patents

Ultrasonic molecular probe and preparation method thereof Download PDF

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CN111150856A
CN111150856A CN201911175627.XA CN201911175627A CN111150856A CN 111150856 A CN111150856 A CN 111150856A CN 201911175627 A CN201911175627 A CN 201911175627A CN 111150856 A CN111150856 A CN 111150856A
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molecular probe
sirna
nps
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黄帅帅
古丽米热·图尔贡
任雨
王雪
翁国斌
祝杭婕
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Medical Community Of Second Hospital Of Yinzhou District Ningbo
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Abstract

The invention relates to an ultrasonic molecular probe, which comprises a liposome nanoparticle shell membrane, liquid fluorocarbon wrapped in the shell membrane and an antibody carried on the surface of the shell membrane, and is characterized in that: the antibody is carbonic anhydrase IX monoclonal antibody, and the surface of the shell membrane also carries siRNA of cyclic adenosine monophosphate response element binding protein, wherein the sequence of the siRNA of the cyclic adenosine monophosphate response element binding protein is SEQ ID NO: 1. the ultrasonic molecular probe provided by the invention is small in size, can penetrate through vascular endothelial gaps and can be used for searching kidney cancer cells in a targeted manner, liquid-gas phase change of liquid fluorocarbon wrapped in the ultrasonic molecular probe can be generated after ultrasonic irradiation, microbubble and cavitation effects are generated, blood flow backscattering is enhanced, an ultrasonic imaging result is improved, and the ultrasonic molecular probe can be specifically combined with target cells, so that the ingestion of target SiCREB with a cancer suppression effect is promoted, and the growth of the kidney cancer cells is suppressed.

Description

Ultrasonic molecular probe and preparation method thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to an ultrasonic molecular probe and a preparation method thereof.
Background
Renal cancer (RCC) is one of the common malignant tumors of the urogenital system, and the morbidity and mortality of the renal cancer are on the rise in recent years, so that the renal cancer brings serious harm to the physical and psychological health of patients. The early diagnosis and effective intervention treatment of RCC are important aiming at the characteristics of high morbidity and mortality, poor treatment effect and the like of RCC.
The clinical existing imaging technology (such as CT, ultrasound, MRI and the like) and laboratory tumor marker screening have certain difficulty in the qualitative diagnosis, prognosis evaluation and transfer reexamination of RCC. In recent years, a molecular imaging technology which is formed by combining a traditional medical imaging technology with modern molecular biology is adopted, through the design of an ultrasonic molecular probe, by means of principles such as enzymatic activation, targeted molecular combination and the like, a multifunctional molecular probe which is used for modifying a target point on the surface and carrying a medicine or a gene is used, the gene expression and the signal transmission process between molecules are visually displayed in an imaging mode, the development characteristics of diseases are better understood clinically, and a new thought and means are provided for early-stage image diagnosis and targeted precise treatment of RCC at the molecular or cell level.
The ultrasound microbubble has better ultrasound imaging effect as a micron-sized ultrasound contrast agent containing gas, is widely applied to diagnosis of various clinical diseases (such as Sonowei SonoVue, the average diameter is about 2.5 μm) at present, and is also used as a carrier capable of carrying anti-tumor drugs or genes for experimental research. However, the micron-scale particle size of conventional ultrasound microbubbles does not allow deep targeting of extravascular tumor tissue through the tumor endothelial cell space. The liposome nanoparticle with a small particle size can smoothly pass through the endothelial cell gap of tumor tissues and can be used as a carrier of antitumor drugs/genes for antitumor treatment, but the liposome nanoparticle also has the following defects: the imaging effect of the common liposome nanoparticles based on the principle of enhancing backscatter imaging in an aggregation form is inferior to that of micron-sized ultrasonic microbubbles.
Liquid fluorocarbon (PFP) induced acoustic droplet phase transition effect (ADV) brings new hope for strong association between liposome nanoparticles and ultrasound microbubbles. The better biological safety of PFP provides possibility for the PFP to be used as a core material of an ultrasonic molecular probe. More importantly, Rapoport et al reported that in addition to temperature, the threshold value (Δ P) for ADV of PFP is related to the difference in internal and external surface tension (σ) of the microspheres, i.e., Laplace pressure, and can be obtained according to the following equation:
ΔP＝Pinside-Poutside＝2σ/r。
as can be seen from the above equation, the ADV threshold is related to the particle size (r) of the microspheres, the larger the particle size, the lower the temperature requirement for ADV to occur, and vice versa. When the particle size of the microspheres reaches 4 mu m and the temperature reaches physiological temperature of 37 ℃, ADV can only occur through the liquid fluorocarbon PFP wrapped in the microspheres. The previous experimental results of the applicant show that PFP is wrapped in liposome nanoparticles and prepared into nano-scale, the ADV temperature of the PFP is obviously improved, and the stability of the microspheres is improved. In addition, in the ADV research, the change of the ultrasound intensity, frequency and pulse mode can affect the threshold of the ADV, wherein the increase of the ultrasound intensity, frequency and time can adjust the ADV temperature down, and the low-frequency focused ultrasound (abbreviated as LIFU) can generate the cavitation effect after the ADV, thereby achieving the therapeutic effect.
However, previous experiments by the applicant show that the common liposome nanoparticles still cannot perform efficient imaging and treatment of diseases, and the content of the liposome nanoparticles reaching target tissues is not high due to the lack of targeting characteristics. It has been reported in literature that, since VHL mutations of RCC patients are lost constant, so that the expression of Carbonic Anhydrase IX (CAIX) downstream thereof is not inhibited, CAIX is highly expressed in ccRCC tissues but not in normal kidney proximal duct epithelial cells, so that CAIX has become a tumor marker of RCC. The CAIX is a member of carbonic anhydrase family, has the capability of catalyzing carbon dioxide and water to reversibly synthesize carbonic acid so as to adjust PH, and has an important function of maintaining the acidic microenvironment of tumor tissues. It has been shown that CAIX mediates tumor growth and metastatic function and contributes to PH regulation, cell proliferation, adhesion, metastasis and invasive processes. The results of early tests of the vaccine modified by the CAIX antibody in treating kidney cancer are introduced in International renal cancer workshop by Alexandra and the like, and the results show that the dendritic cell-CAIX vaccine transduced by the granulocyte-macrophage colony stimulating factor fusion gene can effectively and safely target cell membranes on the surface of kidney cancer tumors and help patients to stabilize the state of the disease. In addition, Lamers et al, using chimeric antibody targeting CAIX to modify T cells, can exert antigen-directed targeting toxicity, with greater resistance to renal cancer. Therefore, the CAIX on the surface of the kidney cancer is an effective specific antigen, and cells or other delivery carriers taking the CAIX as a target are expected to become a new anti-kidney cancer drug transport tool.
The renal cancer is not sensitive to radiotherapy and chemotherapy, so the gene therapy is particularly important in the process of inhibiting the occurrence and development of the renal cancer. At present, tumor targeted therapy plays an increasingly important role in tumor therapy by virtue of specificity and targeting property, and becomes a main attack direction of tumor therapy. The targeted therapy is to design a corresponding therapeutic drug aiming at a well-defined carcinogenic site (the site can be a protein molecule in a tumor cell or a gene fragment) on a cellular molecular level, the drug enters into the body and specifically selects the carcinogenic site to combine and act, so that the tumor cell is specifically killed without affecting normal tissue cells around the tumor, so the molecular targeted therapy is also called biological missile, and therefore, in order to better diagnose the kidney cancer at an early stage and perform effective intervention therapy, a new target and a new means for treating the kidney cancer are needed to be found.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide a new ultrasound molecular probe for molecular targeted therapy of kidney cancer in view of the above technical status.
The second technical problem to be solved by the present invention is to provide a method for preparing an ultrasonic molecular probe in view of the above technical situation.
The first technical solution adopted by the present invention to solve the above technical problems is: an ultrasonic molecular probe comprises a liposome nanoparticle shell membrane, liquid fluorocarbon wrapped in the shell membrane and an antibody carried on the surface of the shell membrane, and is characterized in that: the antibody is carbonic anhydrase IX monoclonal antibody, and the surface of the shell membrane also carries siRNA of cyclic adenosine monophosphate response element binding protein, wherein the sequence of the siRNA of the cyclic adenosine monophosphate response element binding protein is SEQ ID NO: 1, specifically:
Figure BDA0002289863130000031
Figure BDA0002289863130000041
cAMP-responsive element binding protein (CREB) is used as an intracellular proto-oncogenic transcription factor, the Ser-133 site of the CREB is phosphorylated by protein kinases such as PKA, MAPK, PKC, CaMKs, CK II and the like, CBP protein is recruited and combined with highly conserved cAMP responsive element near a target gene promoter, acetylation of the cAMP responsive element is induced, and the transcription function of the target gene is activated and started. The applicant finds in earlier studies that the expression level of CREB in cervical cancer, renal cancer and lymphoma is significantly higher than that of paracarcinoma tissues, and the CREB participates in regulation of cell cycle, apoptosis and invasion related protein expression, so as to influence the development process of cells, and moreover, the siRNA of the cAMP binding protein transfected by a liposome method can significantly inhibit the development of renal cancer cells by reducing the expression of the cAMP binding protein.
The liposome nanoparticle is a novel assembly with a lipid shell and a nanoparticle core, which is formed by self-assembling lipid vesicles with a certain concentration and nanoparticles dispersed in an aqueous solution. The liquid fluorocarbon is wrapped by the liposome nanoparticles, so that the ultrasonic molecular probe can penetrate through a vascular endothelial gap, the carbonic anhydrase IX monoclonal antibody is carried on the shell membrane, the ultrasonic molecular probe can target and search the antigen of the carbonic anhydrase IX monoclonal antibody highly expressed on the surface of a renal cancer cell membrane after penetrating through the vascular endothelial gap, the liquid fluorocarbon is subjected to liquid-gas phase change after ultrasonic irradiation to generate microbubbles and cavitation effect, the blood flow backscattering is enhanced, the ultrasonic imaging result is improved, the permeability of target cells can be increased, the siRNA transfection of the cyclic adenosine monophosphate reaction element binding protein carried on the surface of the liposome nanoparticles is promoted, the occurrence and development of renal cancer cells are inhibited through reducing the expression of the cyclic adenosine monophosphate reaction element binding protein in renal cancer tissues, and the treatment effect is achieved.
As the first design of the siRNA sequence of the cyclic adenosine monophosphate response element binding protein, the sense strand sequence of the siRNA of the cyclic adenosine monophosphate response element binding protein is as follows: 5'-GUCUCCACAAGUCCAAACATT-3', the antisense strand sequence is: 5'-GUCUCCACAAGUCCAAACATT-3' are provided.
As a second design of the sequence of the siRNA of the cyclic amp response element binding protein, the sense strand sequence of the siRNA of the cyclic amp response element binding protein is: 5'-GGCAGACAGUUCAAGUCCAUG-3', the antisense strand sequence is: 5'-UGGACUUGAACUGUCUGCCCA-3' are provided.
As a third design of the sequence of the siRNA of the cyclic amp response element binding protein, the sense strand sequence of the siRNA of the cyclic amp response element binding protein is: 5'-UAAUUCCUUCAAUACCAUGCU-3', the antisense strand sequence is: 5'-CAUGGUAUUGAAGGAAUUAGA-3' are provided.
In order to facilitate the ultrasonic molecular probe to penetrate through a tiny blood vessel and deeply target the extravascular tumor tissue, the particle size range of the ultrasonic molecular probe is 220-450 nm. At present, sononovacin used in clinic has the average diameter of about 2.5 μm, the particle size of the ultrasound microbubble is too large to pass through the tumor endothelial cell gap and is difficult to deeply target the extravascular tumor tissue, and the particle size of the ultrasound molecular probe in the invention is smaller.
The second technical solution adopted by the present invention to solve the above technical problems is: a method for preparing the ultrasonic molecular probe is characterized by comprising the following steps,
the method comprises the following steps: preparing a liposome nanoparticle shell membrane;
step two: adding liquid fluorocarbon into the liposome nanoparticle shell membrane obtained in the step one to obtain liposome nanoparticles with liquid fluorocarbon wrapped inside;
step three: loading siRNA of the cyclic adenosine monophosphate response element binding protein on the surface of the liposome nanoparticle which is obtained in the step two and is internally wrapped with liquid fluorocarbon to obtain the liposome nanoparticle of which the surface is carried with the siRNA of the cyclic adenosine monophosphate response element binding protein;
step four: and (3) carrying out amide reaction on the liposome nanoparticles of the siRNA carrying the cyclic adenosine monophosphate response element binding protein on the surface obtained in the step three and the carbonic anhydrase IX monoclonal antibody to obtain the ultrasonic molecular probe.
Further design, the step of preparing the liposome nanoparticle shell membrane in the step one comprises the following steps:
a. weighing 10mg of dipalmitoylphosphatidylcholine, 3-4mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000-amino cross-linked substance and 2-4mg of 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol into a round-bottomed flask;
b. adding 4-6mL of chloroform and 1-2mL of methanol, mixing and dissolving;
c. evaporating in rotary water bath at 50-80 deg.C for 0.5-1 hr to obtain uniform thin layer of liposome nanoparticles.
Further designed, in step a of preparing the liposome nanoparticle shell membrane in step one, the mass of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino cross-linked complex and the mass of 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol were 4mg and 3mg, respectively.
Further design, the step of preparing the liposome nanoparticle with the liquid fluorocarbon wrapped inside in the step two comprises the following steps:
a. adding 2-3mL of double distilled water into the liposome nanoparticle thin layer obtained in the step one to obtain a primary emulsion;
b. precooling the primary emulsion for 1-2min by using an ice water bath, setting acoustic-shock parameters, wherein the acoustic-shock frequency is started and stopped at an interval of 5s, the acoustic-shock power is 125W, and acoustic-shock emulsification is carried out by adopting a multi-acoustic-shock mode, the total acoustic-shock duration is 9-11min, and liquid fluorocarbon with the total amount of 150-;
c. centrifuging, collecting the precipitate, and obtaining the liposome nanoparticles wrapped with liquid fluorocarbon.
In order to make the effect of coating the liquid fluorocarbon by the liposome nanoparticles better, in the step a of preparing the liposome nanoparticles coated with the liquid fluorocarbon inside in the step two, the total amount of the added liquid fluorocarbon is 200 μ L.
In order to enable the effect of wrapping liquid fluorocarbon by the liposome nanoparticles to be better, in the step b of preparing the liposome nanoparticles wrapped with the liquid fluorocarbon inside in the step two, the acoustic shock emulsification is carried out twice, the first acoustic shock time is 5-6min, the second acoustic shock time is 4-5min, and the liquid fluorocarbon is added between the first acoustic shock and the second acoustic shock.
Further designing, the step of preparing the liposome nanoparticle carrying siRNA with cyclic adenosine monophosphate response element binding protein on the surface in the third step is as follows:
a. taking siRNA of the liposome nanoparticle which is obtained in the second step and is internally wrapped with liquid fluorocarbon and the cyclic adenosine monophosphate response element binding protein with the concentration of 0.3-0.7 mu g/mu L, wherein the concentration of the siRNA is 3.5 x 10^8-4.5 x 10^ 8/mL, and the siRNA comprises the following components in percentage by volume: 1-5: 2, the positive potential of the liposome nanoparticle internally wrapped with liquid fluorocarbon is equal to the negative potential of siRNA of the cyclic adenosine monophosphate response element binding protein;
b. centrifuging, washing and collecting the precipitate to obtain the liposome nanoparticle of siRNA carrying cyclic adenosine monophosphate response element binding protein on the surface.
Further design, the step of preparing the ultrasonic molecular probe in the fourth step comprises the following steps:
a. mixing 1-3mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution with the concentration of 0.4-0.5mol/L and 1-3mL of N-hydroxysuccinimide solution with the concentration of 0.1-0.2mol/L, and adding 10-30 mu g of carbonic anhydrase IX monoclonal antibody, wherein the pH values of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and the N-hydroxysuccinimide solution are both 5.5-6.0;
b. adjusting the pH value to 8.0-8.4;
c. adding liposome nanoparticles of siRNA carrying cyclic adenosine monophosphate reaction element binding protein on the surface, which are obtained in the third step and have the amount of substances such as carbonic anhydrase IX monoclonal antibody and the like, and reacting for 20-60 min;
d. centrifuging and rinsing to obtain the ultrasonic molecular probe.
In the invention, the probe is abbreviated as CAIX-sicEB-PFP-NPS for the ultrasonic molecular probe, the shell membrane of liposome nanoparticle is abbreviated as NPS, the shell membrane of liquid fluorocarbon is abbreviated as PFP, the carbonic anhydrase IX is abbreviated as CAIX, the siRNA of cyclic adenosine monophosphate reaction element conjugated protein is abbreviated as sCREB-NPB, the liposome nanoparticle coated with liquid fluorocarbon internally, the siRNA nanoparticle carrying the siRNA of cyclic adenosine monophosphate reaction element conjugated protein on the surface is abbreviated as sCREB-PFP-NPS, the DPPC of dipalmitoylphosphatidylcholine is abbreviated as DPPC, the DSPE-PEG2000-NH2 for the distearoylphosphatidylethanolamine-polyethylene glycol 2000-amino cross-linked substance is abbreviated as DSPE-PEG2000-NH2, 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol, EDC of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, and NHS for the N-hydroxysuccinimide.
Compared with the prior art, the invention has the advantages that: the ultrasonic molecular probe is nanoscale, has small volume, can penetrate through vascular endothelial gaps and can target and search kidney cancer cells, and can also enable liquid-gas phase change of liquid fluorocarbon wrapped in the ultrasonic molecular probe after ultrasonic irradiation to generate microbubble and cavitation effects, enhance the backscattering of blood flow, improve the ultrasonic imaging result, increase the permeability of target cells, thereby promoting the transfection of the siCREB carried on the surface of liposome nanoparticles with anti-tumor effect and inhibiting the kidney cancer; the invention also provides a method for preparing the ultrasonic molecular probe, which has the characteristic of simple and convenient operation.
Drawings
FIG. 1 is a schematic structural diagram of an ultrasonic molecular probe in the present invention;
FIG. 2 is a light mirror diagram of the ultrasonic molecular probe in example 1 of the present invention;
FIG. 3 is a diagram showing a potential analysis of the ultrasonic molecular probe in example 1 of the present invention;
FIG. 4 is a graph showing particle size analysis of the ultrasonic molecular probe in example 1 of the present invention;
FIG. 5 is a graph of flow cytometry analysis of siCREB-PFP-NPS carrying siCREB (PFP-NPS) in example 1 of the present invention;
FIG. 6 is a diagram of the flow cytometric analysis of siCREB-PFP-NPS in example 1 of the present invention;
FIG. 7 is a diagram of the flow cytometry analysis of CAIX-sicEB-PFP-NPS carrying CAIX pre (sicEB-PFP-NPS) in example 1 of the present invention;
FIG. 8 is a diagram of the flow cytometry analysis of the ultrasonic molecular probe CAIX-sicEB-PFP-NPS in example 1 of the present invention;
FIG. 9 is a diagram showing the thermal induced phase transition of the ultrasonic molecular probe in example 1 of the present invention at 50 ℃;
FIG. 10 is the diagram of the phase change caused by sound of the ultrasound molecular probe in the US mode and the CEUS mode in the embodiment 1 of the present invention;
FIG. 11 is a diagram showing the specific binding of the ultrasonic molecular probe to 786-O cells in example 1 of the present invention;
FIG. 12 shows the effect of the ultrasound molecular probe of example 1 on 786-O cell proliferation after low-frequency focused ultrasound.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
As shown in FIG. 1, the CAIX-siCREB-PFP-NPS in the present example includes NPS, PFP encapsulated in NPS and NPS surface carried antibody, and is characterized in that: the antibody is CAIX mab and the NPS surface also carries siCREB, wherein the sequence of siCREB is SEQ ID NO: 1.
the sense strand sequence of the siCREB sequence in this example is: 5'-GGCAGACAGUUCAAGUCCAUG-3', the antisense strand sequence is: 5'-UGGACUUGAACUGUCUGCCCA-3' are provided.
The method for preparing the ultrasonic molecular probe in the embodiment comprises the following steps,
the method comprises the following steps: preparing NPS;
step two: adding PFP into the NPS obtained in the step one to obtain PFP-NPS;
step three: loading siCREB on the PFP-NPS surface obtained in the step two to obtain siCREB-PFP-NPS;
step four: and (3) carrying out amide reaction on the siCREB-PFP-NPS obtained in the step three and the CAIX monoclonal antibody to obtain the CAIX-siCREB-PFP-NPS.
Wherein the step of preparing the NPS in the step one is as follows:
a. weighing: weighing 10mg of DPPC and 4mg of DSPE-PEG2000-NH23mg of DC-chol, and placing the mixture in a round-bottom flask;
b. dissolving an organic solvent: adding 5mL of chloroform and 1mL of methanol into a round-bottom flask, and gently shaking up;
c. film forming: connecting with a rotary evaporator, heating and evaporating at 60 deg.C in water bath, and forming uniform NPS thin layer after 45 min.
The step of preparing PFP-NPS in the second step is as follows:
a. water and: taking out the round-bottom flask, and adding 2mL of double distilled water into the NPS thin layer obtained in the step one c to obtain a primary emulsion;
b. acoustic vibration: transferring the primary emulsion in the step two a into a centrifuge tube, then placing the centrifuge tube in an ice-water bath environment for precooling for 2min, inserting a probe of a sound vibration instrument into the liquid level of the primary emulsion, setting first sound vibration parameters (frequency: starting and stopping at intervals of 5s, power: 125W, time: 6min), adding 200uL PFP after the first sound vibration is finished, setting second sound vibration parameters (frequency: starting and stopping at intervals of 5s, power: 125W, time: 4min), and obtaining white emulsified liquid after the first sound vibration is finished;
c. centrifuging: centrifuging for 5min at 8000rpm and 4 deg.C, and collecting precipitate to obtain PFP-NPS.
The step of preparing the siCREB-PFP-NPS in the step three is as follows:
a. preparing a siCREB: the method is characterized in that the method entrusts Shanghai Jima gene biology company to synthesize a SiCREB sequence, and a sense strand sequence of the SiCREB sequence is as follows: 5'-GGCAGACAGUUCAAGUCCAUG-3', the antisense strand sequence is: 5'-UGGACUUGAACUGUCUGCCCA-3' are provided.
b. PFP-NPS reacted with siCREB: diluting the PFP-NPS obtained in the step two c to the concentration of 4.0 x 10^ 8/mL, diluting the sicREB obtained in the step three a to 0.5 mu g/mu L, uniformly mixing 200 mu L of PFP-NPS and 60 mu L of sicREB, and standing for 30min at the temperature of 4 ℃;
c. centrifuging: centrifuging for 3min at the rotation speed of 400rpm, and obtaining the precipitate, namely the siCREB-PFP-NPS.
The step of preparing CAIX-sicEB-PFP-NPS in the fourth step is as follows:
a. dissolving: mixing 1mL of EDC solution with the concentration of 0.4mol/L and 1mL of NHS solution with the concentration of 0.1mol/L, adding 20 mu g of CAIX antibody, and mixing and reacting in a shaking table for 1h, wherein the pH values of the EDC solution and the NHS solution are both 5.5;
b. adjusting the pH value: regulating the pH value of the mixed solution to 8.0 by using 1mol/L NaOH solution;
c. amide reaction: adding the siCREB-PFP-NPS obtained in the third step with the amount of substances such as CAIX antibodies and the like, and continuously reacting for 30min in a shaking table;
d. centrifuging: centrifuging at 300rpm for 5min, rinsing with PBS solution, repeating for 2 times, and collecting the upper layer nanoparticle solution to obtain CAIX-sicEB-PFP-NPS.
The structure and the characteristic features of the ultrasonic molecular probe CAIX-sicEB-PFP-NPS prepared in the embodiment are as follows: (1) as shown in FIG. 2, the CAIX-siCREB-PFP-NPS obtained in this example was examined by a general optical microscope, and the CAIX-siCREB-PFP-NPS had a high concentration under the optical microscope and had a round shape, uniform size, good dispersibility and no aggregation.
(2) As shown in FIG. 3, when the CAIX-siCREB-PFP-NPS obtained in this example was examined by Malvern laser particle size analyzer, the particle size of CAIX-siCREB-PFP-NPS was 289.7. + -. 93.1nm, which was smaller than sononovadin (with an average diameter of about 2.5 μm) used in clinical practice, and was favorable for penetrating through small blood vessels.
(3) As shown in FIG. 4, the potential of the CAIX-siCREB-PFP-NPS obtained in this example was analyzed, and the potential of the CAIX-siCREB-PFP-NPS was-0.23. + -. 0.11mV, which is close to neutral charge;
(4) as shown in fig. 5-6, the siCREB-PFP-NPS obtained in this example was analyzed by flow cytometry for CY-3 fluorescence intensity detection, and fig. 5 is PFP-NPS, which is taken as a control and combined with fig. 6, so that the carrying amount of siCREB in nanoparticles is 91.18% ± 2.41%.
(5) As shown in FIGS. 7-8, FITC fluorescence intensity detection analysis was performed on the CAIX-sicEB-PFP-NPS obtained in this example by flow cytometry, and as a control, the CAIX-sicEB-PFP-NPS obtained in this example was connected to FIG. 8, whereby the CAIX-attachment rate in the nanoparticles was 63.98% + -3.11%.
(6) Thermotropic phase transition study of the CAIX-sicEB-PFP-NPS obtained in this example
As shown in fig. 9, in the implementation process of the CAIX-siCREB-PFP-NPS thermal phase transition experiment, the CAIX-siCREB-PFP-NPS is placed in a heated glass plate, and when the temperature is at normal temperature, the phase transition of the nanoparticles does not occur; when the temperature is heated to 50 ℃, the volume of the nanoparticles is larger and larger, and the number of the nanoparticles subjected to phase change is increased.
(7) The CAIX-sicEB-PFP-NPS obtained in this example was subjected to a sonogenic transformation study
As shown in fig. 10, in the implementation of the CAIX-siCREB-PFP-NPS phase-change acoustic experiment, the CAIX-siCREB-PFP-NPS is placed in a gel model, and ultrasonic intensities of 0w, 2.0w and 4.0w are respectively applied, and the irradiation is performed for 1min and 3min, and it is found that when the ultrasonic irradiation is 2.0w, the US and CEUS echoes of the nanoparticles are gradually enhanced, and 4.0w is sometimes weakened.
In conclusion, the ultrasonic molecular probe CAIX-siCREB-PFP-NPS prepared in the embodiment has the characteristics of small particle size, strong phase change capability and high silicon-loaded reb amount.
(8) In vitro renal cancer 786-O cell phagocytosis assay of CAIX-sicEB-PFP-NPS obtained in this example
Preparing materials of PFP-NPS group, siCREB-PFP-NPS group and CAIX-siCREB-PFP-NPS group, carrying out Dil staining in the preparation process of each group of materials, adding 786-O cells of kidney cancer into each group, treating for 1h, carrying out DAPI staining for 3min, and observing by a fluorescence microscope.
As shown in FIG. 11, the Dil signals of the PFP-NPS group and the siCREB-PFP-NPS group are weak, and the relative fluorescence intensities are 1.01 + -0.11 and 1.33 + -0.19 respectively, which shows that the renal cancer 786-O cells phagocytose the molecular probes of the PFP-NPS group and the siCREB-PFP-NPS group are less, while the Dil signals of the CAIX-siCREB-PFP-NPS group are strong, and the relative fluorescence intensity is 3.36 + -0.17, which shows that the renal cancer 786-O cells phagocytose the molecular probes of the CAIX-siCREB-PFP-NPS group more, and have significant difference compared with the other two groups. Therefore, the targeting and targeting binding capacity of the CAIX-siCREB-PFP-NPS group on the cells is obviously higher than that of the PFP-NPS group and the siCREB-PFP-NPS group; therefore, the ultrasound molecular probe CAIX-siCREB-PFP-NPS prepared in the embodiment can target and specifically bind to the surface of the cell membrane of the kidney cancer 786-O.
(9) In vitro renal cancer 786-O cell assay study of CAIX-sicEB-PFP-NPS obtained in this example
Renal cancer 786-O cells were added to the placebo group, the PFP-NPS group, the siCREB-treated group, the CAIX-treated group, the siCREB-PFP-NPS group, and the CAIX-siCREB-PFP-NPS group, respectively, and after 24 hours of treatment, the proliferation potency of the anti-renal cancer 786-O cells was examined by the MTT method.
As shown in fig. 12, there was no significant difference in cytotoxicity of the placebo, PFP-NPS, siCREB-treated, CAIX-treated and siCREB-PFP-NPS-treated groups, but the CAIX-siCREB-PFP-NPS group was significantly downregulated in its proliferative capacity compared to the placebo group; therefore, the ultrasonic molecular probe CAIX-siCREB-PFP-NPS prepared in the embodiment can obviously inhibit the proliferation capacity of renal cancer 786-O cells.
Therefore, the ultrasonic molecular probe CAIX-siCREB-PFP-NPS prepared by the embodiment has good dispersity, uniform size and obvious targeting property, and the successful preparation of the ultrasonic molecular probe lays a good foundation for the next accurate treatment of tumor cells.
Example 2
As shown in FIG. 1, the CAIX-siCREB-PFP-NPS in the present example includes NPS, PFP encapsulated in NPS and NPS surface carried antibody, and is characterized in that: the antibody is CAIX mab and the NPS surface also carries siCREB, wherein the sequence of siCREB is SEQ ID NO: 1. the preparation process is essentially the same as that of example 1, with the following differences: the sense strand sequence of the siCREB sequence in this example is: 5'-GUCUCCACAAGUCCAAACATT-3', the antisense strand sequence is: 5'-GUCUCCACAAGUCCAAACATT-3' are provided.
The preparation method of the ultrasonic molecular probe in the embodiment comprises the following steps,
the method comprises the following steps: preparing NPS;
step two: adding PFP into the NPS obtained in the step one to obtain PFP-NPS;
step three: loading siCREB on the PFP-NPS surface obtained in the step two to obtain siCREB-PFP-NPS;
step four: and (3) carrying out amide reaction on the siCREB-PFP-NPS obtained in the step three and the CAIX monoclonal antibody to obtain the CAIX-siCREB-PFP-NPS.
Wherein the step of preparing the NPS in the step one is as follows:
a. weighing: weighing 10mg of DPPC and 3mg of DSPE-PEG2000-NH22mg of DC-chol, placing in a round-bottom flask;
b. dissolving an organic solvent: adding 4mL of chloroform and 1.5mL of methanol into a round-bottom flask, and gently shaking up;
c. film forming: connecting with a rotary evaporator, heating and evaporating at 80 deg.C in water bath, and forming uniform NPS thin layer after 0.5 hr.
The step of preparing PFP-NPS in the second step is as follows:
a. water and: taking out the round-bottom flask, and adding 2.5mL of double distilled water into the NPS thin layer obtained in the step one c to obtain a primary emulsion;
b. acoustic vibration: absorbing the primary emulsion in the step two a into a centrifuge tube, precooling for 2min in an ice-water bath, inserting a probe of a sound vibration instrument below the liquid level of the primary emulsion, setting first sound vibration parameters (frequency: starting and stopping at intervals of 5s, power: 125W, time: 5min), adding 150 mu L PFP after the first sound vibration is finished, setting second sound vibration parameters (frequency: starting and stopping at intervals of 5s, power: 125W, time: 5min), and obtaining white emulsified liquid after the first sound vibration is finished;
c. centrifuging: centrifuging at 9000rpm and 4 deg.C for 5min, and collecting precipitate to obtain PFP-NPS.
The step of preparing the siCREB-PFP-NPS in the step three is as follows:
a. preparing a siCREB: the method is characterized in that the method entrusts Shanghai Jima gene biology company to synthesize a SiCREB sequence, and a sense strand sequence of the SiCREB sequence is as follows: 5'-GUCUCCACAAGUCCAAACATT-3', the antisense strand sequence is: 5'-GUCUCCACAAGUCCAAACATT-3' are provided.
b. PFP-NPS reacted with siCREB: diluting the PFP-NPS obtained in the step two c to the concentration of 4.5 x 10^ 8/mL, diluting the sicREB obtained in the step three a to 0.7 mu g/mu L, uniformly mixing 200 mu L of PFP-NPS and 80 mu L of sicREB, and standing for 30min at the temperature of 4 ℃;
c. centrifuging: centrifuging for 3min at the rotating speed of 400rpm, and obtaining the precipitate, namely the siCREB-PFP-NPS.
The step of preparing CAIX-sicEB-PFP-NPS in the fourth step is as follows:
a. dissolving: mixing 2mL of EDC solution with the concentration of 0.4mol/L and 2mL of NHS solution with the concentration of 0.1mol/L, adding 10 mu g of CAIX antibody, and mixing and reacting in a shaking table for 1h, wherein the pH values of the EDC solution and the NHS solution are both 5.5;
b. adjusting the pH value: regulating the pH value of the mixed solution to 8.2 by using 1mol/L NaOH solution;
c. amide reaction: adding the siCREB-PFP-NPS obtained in the third step with the amount of substances such as CAIX antibodies and the like, and continuously reacting for 20min in a shaking table;
d. centrifuging: centrifuging at 300rpm for 5min, rinsing with PBS solution, repeating for 2 times, and collecting the upper layer nanoparticle solution to obtain CAIX-sicEB-PFP-NPS.
The CAIX-siCREB-PFP-NPS obtained in the embodiment is detected by a common optical microscope, and the CAIX-siCREB-PFP-NPS has higher concentration, circular shape, uniform size, good dispersibility and no aggregation under the optical microscope; when the CAIX-siCREB-PFP-NPS obtained in the embodiment is detected by a Malvern laser particle size analyzer, the particle size of the CAIX-siCREB-PFP-NPS is 347.2 +/-105.4 nm, and the particle size is smaller than that of sononovalvir (the average diameter of the sononovalvir is about 2.5 mu m) used clinically, so that the CAIX-siCREB-PFP-NPS is favorable for penetrating a micro blood vessel; the potential analysis of the CAIX-siCREB-PFP-NPS obtained in the example shows that the potential of the CAIX-siCREB-PFP-NPS is-0.20 +/-0.08 mV and is close to neutral charge; the CY-3 fluorescence intensity detection analysis of the siCREB-PFP-NPS obtained in this example was performed by flow cytometry, and the carrying amount of the siCREB in the nanoparticles was 91.02% ± 2.13%; FITC fluorescence intensity detection analysis of the CAIX-sicEB-PFP-NPS obtained in the example by flow cytometry shows that the connection rate of CAIX in the nanoparticles is 60.39% + -4.68%.
Example 3
As shown in FIG. 1, the CAIX-siCREB-PFP-NPS in the present example includes NPS, PFP encapsulated in NPS and NPS surface carried antibody, and is characterized in that: the antibody is CAIX mab and the NPS surface also carries siCREB, wherein the sequence of siCREB is SEQ ID NO: 1, substantially the same as the preparation of example 1, except that: the sense strand sequence of the siCREB sequence in this example is: 5'-UAAUUCCUUCAAUACCAUGCU-3', the antisense strand sequence is: 5'-CAUGGUAUUGAAGGAAUUAGA-3' are provided.
The method for preparing the ultrasonic molecular probe in the embodiment comprises the following steps,
the method comprises the following steps: preparing NPS;
step two: adding PFP into the NPS obtained in the step one to obtain PFP-NPS;
step three: loading siCREB on the PFP-NPS surface obtained in the step two to obtain siCREB-PFP-NPS;
step four: and (3) carrying out amide reaction on the siCREB-PFP-NPS obtained in the step three and the CAIX monoclonal antibody to obtain the CAIX-siCREB-PFP-NPS.
Wherein the step of preparing the NPS in the step one is as follows:
a. weighing: weighing 10mg of DPPC, 4mg of DSPE-PEG2000-NH2 and 4mg of DC-chol, and placing the mixture in a round-bottom flask;
b. dissolving an organic solvent: adding 6mL of chloroform and 2mL of methanol into a round-bottom flask, and gently shaking up;
c. film forming: connecting with a rotary evaporator, heating and evaporating at 50 ℃ by a water bath method, and forming a uniform NPS thin layer after 1 h.
The step of preparing PFP-NPS in the second step is as follows:
a. water and: taking out the round-bottom flask, and adding 3mL of double distilled water into the NPS thin layer obtained in the step one c to obtain a primary emulsion;
b. acoustic vibration: absorbing the primary emulsion in the step two a into a centrifuge tube, precooling for 1min in an ice-water bath, inserting a probe of a sound vibration instrument below the liquid level of the primary emulsion, setting first sound vibration parameters (frequency: starting and stopping at intervals of 5s, power: 125W, time: 6min), adding 175 mu L PFP after the first sound vibration is finished, setting second sound vibration parameters (frequency: starting and stopping at intervals of 5s, power: 125W, time: 5min), and obtaining white emulsified liquid after sound vibration;
c. centrifuging: centrifuging for 5min at 8500rpm and 4 deg.C, and collecting precipitate to obtain PFP-NPS.
The step of preparing the siCREB-PFP-NPS in the step three is as follows:
a. preparing a siCREB: the method is characterized in that the method entrusts Shanghai Jima gene biology company to synthesize a SiCREB sequence, and a sense strand sequence of the SiCREB sequence is as follows: 5'-UAAUUCCUUCAAUACCAUGCU-3', the antisense strand sequence is: 5'-CAUGGUAUUGAAGGAAUUAGA-3' are provided.
b. PFP-NPS reacted with siCREB: diluting the PFP-NPS obtained in the step two c to the concentration of 3.5 x 10^ 8/mL, diluting the sicREB obtained in the step three a to 0.3 mu g/mu L, uniformly mixing 200 mu L of PFP-NPS and 40 mu L of sicREB, and standing for 30min at the temperature of 4 ℃;
c. centrifuging: centrifuging for 3min at the rotating speed of 400rpm, and obtaining the precipitate, namely the siCREB-PFP-NPS.
The step of preparing CAIX-sicEB-PFP-NPS in the fourth step is as follows:
a. dissolving: mixing 3mL of EDC solution with the concentration of 0.5mol/L and 3mL of NHS solution with the concentration of 0.2mol/L, adding 30 mu g of CAIX antibody, and mixing and reacting in a shaking table for 1h, wherein the pH values of the EDC solution and the NHS solution are both 6.0;
b. adjusting the pH value: regulating the pH value of the mixed solution to 8.4 by using 1mol/L NaOH solution;
c. amide reaction: adding the siCREB-PFP-NPS obtained in the step three with the amount of substances such as CAIX antibodies and the like, and continuously reacting for 1h in a shaking table;
d. centrifuging: centrifuging at 300rpm for 5min, rinsing with PBS solution, repeating for 3 times, and collecting the upper layer nanoparticle solution to obtain CAIX-sicEB-PFP-NPS.
The CAIX-siCREB-PFP-NPS obtained in the embodiment is detected by a common optical microscope, and the CAIX-siCREB-PFP-NPS has higher concentration, circular shape, uniform size, good dispersibility and no aggregation under the optical microscope; when the CAIX-siCREB-PFP-NPS obtained in the embodiment is detected by a Malvern laser particle size analyzer, the particle size of the CAIX-siCREB-PFP-NPS is 302.5 +/-84.1 nm, and compared with sononovalvir (the average diameter of the sononovalvir is about 2.5 mu m), the particle size is smaller, so that the CAIX-siCREB-PFP-NPS is favorable for penetrating a tiny blood vessel; the potential analysis of the CAIX-siCREB-PFP-NPS obtained in the example shows that the potential of the CAIX-siCREB-PFP-NPS is-0.24 +/-0.16 mV and is close to neutral charge; the CY-3 fluorescence intensity detection analysis of the siCREB-PFP-NPS obtained in this example was performed by flow cytometry, and the carrying amount of the siCREB in the nanoparticles was 88.53% ± 1.75%; FITC fluorescence intensity detection analysis of the CAIX-sicEB-PFP-NPS obtained in the example by flow cytometry shows that the connection rate of the CAIX in the nanoparticles is 62.73% +/-3.45%.
Sequence listing
<110> Ningbo city \37150, second hospital medical community in State area
<120> ultrasonic molecular probe and preparation method thereof
<130>Not published yet
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<170>PatentIn version 3.5
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Claims (10)

1. An ultrasonic molecular probe comprises a liposome nanoparticle shell membrane, liquid fluorocarbon wrapped in the shell membrane and an antibody carried on the surface of the shell membrane, and is characterized in that: the antibody is carbonic anhydrase IX monoclonal antibody, and the surface of the shell membrane also carries siRNA of cyclic adenosine monophosphate response element binding protein, wherein the sequence of the siRNA of the cyclic adenosine monophosphate response element binding protein is SEQ ID NO: 1.
2. the ultrasound molecular probe of claim 1, wherein the sense strand sequence of the siRNA of the cAMP response element binding protein is: 5'-GUCUCCACAAGUCCAAACATT-3', the antisense strand sequence is: 5'-GUCUCCACAAGUCCAAACATT-3' are provided.
3. The ultrasound molecular probe of claim 1, wherein the sense strand sequence of the siRNA of the cAMP response element binding protein is: 5'-GGCAGACAGUUCAAGUCCAUG-3', the antisense strand sequence is: 5'-UGGACUUGAACUGUCUGCCCA-3' are provided.
4. The ultrasound molecular probe of claim 1, wherein the sense strand sequence of the siRNA of the cAMP response element binding protein is: 5'-UAAUUCCUUCAAUACCAUGCU-3', the antisense strand sequence is: 5'-CAUGGUAUUGAAGGAAUUAGA-3' are provided.
5. A method of preparing an ultrasonic molecular probe according to any one of claims 1 to 4, comprising the steps of,
the method comprises the following steps: preparing a liposome nanoparticle shell membrane;
step two: adding liquid fluorocarbon into the liposome nanoparticle shell membrane obtained in the step one to obtain liposome nanoparticles with liquid fluorocarbon wrapped inside;
step three: loading siRNA of the cyclic adenosine monophosphate response element binding protein on the surface of the liposome nanoparticle which is obtained in the step two and is internally wrapped with liquid fluorocarbon to obtain the liposome nanoparticle of which the surface is carried with the siRNA of the cyclic adenosine monophosphate response element binding protein;
step four: and (3) carrying out amide reaction on the liposome nanoparticles of the siRNA carrying the cyclic adenosine monophosphate response element binding protein on the surface obtained in the step three and the carbonic anhydrase IX monoclonal antibody to obtain the ultrasonic molecular probe.
6. The method for preparing an ultrasonic molecular probe according to claim 5, wherein the step of preparing the liposome nanoparticle shell membrane in the first step comprises:
a. weighing 10mg of dipalmitoylphosphatidylcholine, 3-4mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000-amino cross-linked substance and 2-4mg of 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol into a round-bottomed flask;
b. adding 4-6mL of chloroform and 1-2mL of methanol, mixing and dissolving;
c. evaporating in rotary water bath at 50-80 deg.C for 0.5-1 hr to obtain uniform thin layer of liposome nanoparticles.
7. The method for preparing an ultrasonic molecular probe according to claim 5, wherein the step of preparing the liposome nanoparticle internally wrapped with liquid fluorocarbon in the second step comprises:
a. adding 2-3mL of double distilled water into the liposome nanoparticle thin layer obtained in the step one to obtain a primary emulsion;
b. precooling the primary emulsion for 1-2min by using an ice water bath, setting acoustic-shock parameters, wherein the acoustic-shock frequency is started and stopped at an interval of 5s, the acoustic-shock power is 125W, and acoustic-shock emulsification is carried out by adopting a multi-acoustic-shock mode, the total acoustic-shock duration is 9-11min, and liquid fluorocarbon with the total amount of 150-;
c. centrifuging, collecting the precipitate, and obtaining the liposome nanoparticles wrapped with liquid fluorocarbon.
8. The method of preparing an ultrasonic molecular probe according to claim 7, characterized in that: in the step b of preparing the liposome nanoparticles wrapped with liquid fluorocarbon inside in the step two, the acoustic shock emulsification is carried out twice, the first acoustic shock time is 5-6min, the second acoustic shock time is 4-5min, and the liquid fluorocarbon is added between the first acoustic shock and the second acoustic shock.
9. The method for preparing an ultrasonic molecular probe according to claim 5, wherein the step of preparing the liposome nanoparticle carrying the siRNA with the cyclic AMP response member binding protein on the surface in the third step comprises the steps of:
a. taking siRNA of the liposome nanoparticle which is obtained in the second step and is internally wrapped with liquid fluorocarbon and the cyclic adenosine monophosphate response element binding protein with the concentration of 0.3-0.7 mu g/mu L, wherein the concentration of the siRNA is 3.5 x 10^8-4.5 x 10^ 8/mL, and the siRNA comprises the following components in percentage by volume: 1-5: 2, mixing;
b. centrifuging, washing and collecting the precipitate to obtain the liposome nanoparticle of siRNA carrying cyclic adenosine monophosphate response element binding protein on the surface.
10. The method for preparing the ultrasonic molecular probe according to claim 5, wherein the step of preparing the ultrasonic molecular probe in the fourth step is:
a. mixing 1-3mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution with the concentration of 0.4-0.5mol/L and 1-3mL of N-hydroxysuccinimide solution with the concentration of 0.1-0.2mol/L, and adding 10-30 mu g of carbonic anhydrase IX monoclonal antibody, wherein the pH values of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and the N-hydroxysuccinimide solution are both 5.5-6.0;
b. adjusting the pH value to 8.0-8.4;
c. adding liposome nanoparticles of siRNA carrying cyclic adenosine monophosphate reaction element binding protein on the surface, which are obtained in the third step and have the amount of substances such as carbonic anhydrase IX monoclonal antibody and the like, and reacting for 20-60 min;
d. centrifuging and rinsing to obtain the ultrasonic molecular probe.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955975A (en) * 2009-07-14 2011-01-26 重庆医科大学 Construction of si RNA expression vector of tissue inhibitor of metalloproteinases (TIMPs) and application in treatment of cirrhosis
CN105561304A (en) * 2014-10-15 2016-05-11 常熟浸大科技有限公司 Toad venom targeted liposome and preparation method and application thereof
CN107648620A (en) * 2017-09-13 2018-02-02 中国人民解放军第三军医大学第附属医院 Carry fit targeted ultrasound nanometer bubbles of CAIX and preparation method thereof
CN108014349A (en) * 2017-12-28 2018-05-11 重庆医科大学附属第二医院 A kind of preparation method and applications for the multi-functional contrast agent for carrying gene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955975A (en) * 2009-07-14 2011-01-26 重庆医科大学 Construction of si RNA expression vector of tissue inhibitor of metalloproteinases (TIMPs) and application in treatment of cirrhosis
CN105561304A (en) * 2014-10-15 2016-05-11 常熟浸大科技有限公司 Toad venom targeted liposome and preparation method and application thereof
CN107648620A (en) * 2017-09-13 2018-02-02 中国人民解放军第三军医大学第附属医院 Carry fit targeted ultrasound nanometer bubbles of CAIX and preparation method thereof
CN108014349A (en) * 2017-12-28 2018-05-11 重庆医科大学附属第二医院 A kind of preparation method and applications for the multi-functional contrast agent for carrying gene

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JIAYING CHEN ET AL: "CREB1 regulates glucose transport of gliona cell line U87 by targeting GLUT1", 《MOL CELL BIOCHEM》 *
XIAOYAN HUANG ET AL: "Cloning and expression of a novel CREB mRNA splice variant in human testis", 《REPRODUCTION》 *
孟胜男等主编: "《药剂学 在线学习版》", 31 January 2016 *
杨峥等主编: "《肿瘤的诊断与综合治疗学 上》", 30 June 2017, 吉林科学技术出版社 *

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