CN109097399A - The expression vector of long-chain non-coding RNA H19, the cell strain for expressing H19 and its application - Google Patents
The expression vector of long-chain non-coding RNA H19, the cell strain for expressing H19 and its application Download PDFInfo
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Abstract
The invention discloses a kind of virus expression carriers, can steadily be overexpressed h19 gene；Meanwhile a kind of application the invention also discloses cell strain that can stablize expression h19 gene and virus expression carrier and cell strain in the drug of preparation treatment malignant tumour.The inventor of the present application discovered that the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse；Meanwhile inventor constructs the K562 cell stablized and be overexpressed H19, is 1 × 10 by density7K562-H19 the and K562-NEG cell of/mL passes through tail vein injection, it will be in 200 μ L to NOD/SCID Mice Body of cell transplantation, living imaging observes the incidence and survival condition of mouse, it is found that the overexpression of H19 inhibits the growth of tumour and extends the time-to-live of mouse.
Description
Technical field
The present invention relates to the treatment method of malignant tumour, the especially expression vectors of long-chain non-coding RNA H19, expression
The cell strain of H19 and its application.
Background technique
Chronic myelocytic leukemia (chronic myelocytic leukemia, CML) is a kind of occurs in multipotency hematopoiesis
Pernicious bone marrow proliferative tumour on stem cell.CML patient often will appear characteristic chromosome translocation t (9:22) (q34:
Q11) (i.e. Ph chromosome), on No. 9 chromosomes 2 transposition of C-ABL proto-oncogene to No. 22 chromosome long arms breaking point gathering
Area (BCR) forms BCR-ABL fusion, and unconventionality expression BCR-ABL protein leads to the sustained activation of tyrosine kinases, and
Cause the signal transduction pathway in downstream to be activated, to adjust the expression of cell factor, it is a large amount of to eventually lead to marrow stem/progenitor cells
Hyperplasia, apoptosis reduce and the decline of the adhesiveness of marrow stromal cell, and a large amount of still immature myelocytes is caused to be discharged into peripheral blood,
Lead to CML.According to the overall development process of disease, clinically its development process is divided into three periods, respectively slowly
Property phase (Chronic phase, CP), accelerated period (Accelerated phase, AP) and rapid change period (Blastic phase,
BP).Chronic phase disease symptoms slightly without specificity, are mainly shown as abdominal distension, abdominal mass, out of strength, low-heat, splenomegaly, see
90% patient, illness degree are different.
With the rapid development of genomic sequencing technique, it has been found that RNA can by 60%~70% genome sequence
Transcribe, but wherein only less than 2% coding protein sequence, remaining is referred to as non-coding RNA.In fact, very
Many years ago, many non-coding RNAs are just found to have a major impact vital movement, such as are related to the ribosomes of protein synthesis
RNA (rRNAs), transfer RNA (tRNA) and small nuclear rna (snRNA), and these non-coding RNAs are commonly known as non-volume of running one's home
Code RNA.Non-coding RNA attracts extensive attention in recent years, these RNA are primarily referred to as the non-coding RNA with life adjustment effect,
The RNA (LncRNA) studied including microRNA and herein.Research has been found that LncRNA refers to that length is greater than 200 nucleotide
And transcribed by RNA polymerase II and lacked or the RNA without open reading frame, it can between gene, enhancer original part,
The antisense strand of gene, the other positions for including sub-district genome.
With deepening continuously to long-chain non-coding RNA research, it is found that it is many important LncRNA has the function of, takes part in
The important biological process of body, the occurrence and development etc. including cell differentiation, aging, increment, apoptosis and tumour.A variety of LncRNA
Such as H19, X chromosome specificity inactive transcription object (Xist) participate in Genomic Imprinting.
Pachnis etc., although can be transcribed by RNA polymerase II, can be sheared simultaneously tailing in discovery h19 genes in 1984,
But its product for transcribing out can not translate into the protein of biological function, but be sent out in the form of non-coding RNA
The effect of waving.Since H19 product length has been more than 200nt, therefore it is defined as LncRNA.H19 and IGF2 exists in Mice Body
In No. seven chromosome, then it is located at 11p15.5 in human body, at a distance of 90kb, this belongs to first pair and is proved to be with the marking the two
The gene of characteristic.H19 is presented as the paternal marking and maternal expression, and IGF2 is then presented as paternal expression and the maternal marking.The two
Marking characteristic all by the methylation differential modified region (DMR) of H19 promoter upstream 4kb or marking regulatory region (ICR)
Regulation.On spore, H19 has highly conserved feature, the high expression within three periods, when being embryonic development respectively
It is several in hetero-organization other than heart and skeletal muscle in entoderm, mesoderm and its tissue that differentiates, but after being born
It does not express, unless expression can be reactivated under three circumstances: when tissue damage reparation and stress situation and tumour occur.
H19 overall length 2.3kb by 35 small open reading frame, and has 5 ' end cap minor structures and the 3 ' ends of similar mRNA
Poly-A tail fawns on structure, theoretically a kind of protein comprising 256 amino acid moleculars of codified, but LncRNA is only
It plays a role in mRNA level in-site, and any protein molecule can not be encoded.It compiles in highly conserved region in first promoter of H19
The miRNA-675 molecule of code can control the growth of placenta in latter half of gestation by adjusting the expression of IGFLR gene.The study found that
H19 largely assembles in human body placenta element and some embryonic tissues, indicates that H19 may rise very in embry ogenesis and growth and development stage
Important role.After fetal birth, H19 expression is lowered, but maintenance basal expression is thrown away in mammary gland, adrenal gland and uterine tissue
Amount.It has been reported that and shows in the nephroblastoma, embryonal rhabdomyosarcoma at present, H19 plays tumor suppressor gene, however many
In solid tumor (including breast cancer, liver cancer, bladder cancer etc.), H19 is an oncogene.This shows that H19 passes through difference in human body
Mechanism participate in body tumour formation and progress.Need further to be ground there are many more unknown link in these different mechanism
Study carefully discovery.
Summary of the invention
Based on the above issues, present inventor participates in the formation of body tumour and the specific machine of progress in research H19
It was unexpectedly observed that the overexpression of H19 can inhibit the growth of tumour when processed, technical solution of the present invention includes following as a result,
Aspect:
In the first aspect, the present invention provides a kind of virus expression carrier, the carrier can express H19.
Preferably, the carrier can be overexpressed H19.
It is highly preferred that the virus is slow virus.
Preferably, CMV promoter and h19 gene segment are contained in the nucleic acid of the carrier.Wherein, CMV promoter comes from
Cytomegalovirus.
Preferably, eGFP genetic fragment is also contained in the nucleic acid of the carrier.
In the second aspect, the present invention provides a kind of cell strain, the cell strain can stablize expression H19.
Preferably, the cell strain is K562 cell strain.
It is highly preferred that containing the viral vectors that can stablize expression H19 in the cell.
Preferably, the virus is slow virus, in the nucleic acid of the slow virus containing CMV promoter, h19 gene segment and
EGFP genetic fragment.
In the third aspect, the present invention provides above-mentioned carriers and/or cell strain in the drug for preparing treatment malignant tumour
In application.Wherein, malignant tumour is preferably leukaemia.
In conclusion the invention has the benefit that
The inventor of the present application discovered that the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.Together
When, inventor constructs the K562 cell stablized and be overexpressed H19, is 1 × 10 by density7The K562-H19 and K562-NEG of/mL
Cell is by tail vein injection, and by 200 μ L to NOD/SCID Mice Body of cell transplantation, living imaging observes the morbidity feelings of mouse
Condition and survival condition find that the overexpression of H19 inhibits the growth of tumour and extends the time-to-live of mouse.
Detailed description of the invention
Fig. 1 is slow virus carrier structure chart；
Fig. 2 is the testing result figure of Normal group and the expression of K562 and KCL-22 cell H19；
Fig. 3 is the selection result figure of the Real-time PCR to H19-SiRNA ordered sequence；
Fig. 4 is the testing result figure of the expression of the H19 in K562-H19LentiV and K562empty LentiV；
Fig. 5 is the colony number testing result figure of K562-H19Lenti and K562-empty LentiV group, and wherein A is
Colony figure (40 ×)；B is opposite colony number；
Fig. 6 is influence result figure of the H19 overexpression to BALB/c nude mouse；Wherein,
After A shows that injection is overexpressed the P210 cell of H19, the size of mouse tumor；
B shows that injection is overexpressed influence to mouse survival after the P210 cell of H19；
After C shows that injection is overexpressed the P210 cell of H19, the statistical result of mouse tumor volume；
Fig. 7 is that NOD/SCID mouse tail vein transplants K562-NEG-LentiV and K562-H19Lenti V, living imaging
Mouse invasion rate is observed, the result figure of mouse survival rate is recorded.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.Unless otherwise instructed, the concentration of the reagent in the application is mass concentration.
1. main agents and experimental material
2.H19-SiRNA sequence and H19 Lentiviral
2.1 H19 SiRNA sequences
2.2 H19 Lentivirals
H19 Lentiviral is implemented in Guangzhou FulenGen Co., Ltd..
H19 Lentiviral: CS-GS3160-LV201；
Empty Lentiviral: CS-NEG-LV-201
3. cell strain
K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences
4. main agents are prepared
4.1 contain the cell culture medium of 10% serum
Australia fetal calf serum is placed in high-pressure sterilizing pot 56 DEG C, is sub-packed in the healthy and free from worry centrifuge tube of 10mL after 30min fire extinguishing,
Serum after packing saves in -20 DEG C of refrigerators.RPMI-1640 culture medium is sub-packed in 200mL wide-mouth bottle, and every bottle of 180mL is put in
It is saved in 4 DEG C of refrigerators.Fetal calf serum 20mL in Australia is added in every 180mL culture medium, prepares so that blood in RPMI-1640 culture medium
Clear content is 10%.
4.2 pH7.2 phosphate buffers (PBS)
Said components are weighed respectively, dissolve the above-mentioned component weighed up using distilled water and are settled to 1000mL, and high pressure is steamed
Vapour sterilizing 30min, takes out, 4 DEG C of preservations.
4.3 prepare 2.7% methylcellulose semisolid culturemedium
2.7g methylcellulose powder is weighed in wide-mouth bottle, 50mL boiling water is added, dissolves while stirring, 30min takes
Out, to its cooling, sterile 2 × 1640 culture medium of 50mL is added in superclean bench, stirring is stored in 4 DEG C until dissolution
Refrigerator is stand-by.
5. key instrument and material
1 K562 cell culture of embodiment
CML cell K562 is inoculated in RPMI-1640 culture medium, the content of serum is 10% in RPMI-1640, will be inoculated with
It is 37 DEG C, CO that good K562 cell, which is placed in temperature,2It is cultivated in the cell incubator that volume fraction is 5%.
The screening of 2 H19-SiRNA ordered sequence of embodiment
2.2.1 H19-SiRNA transfection K 562 cell
(1) experimental group are as follows: blank control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19-
SiRNA-03 group, the concentration of SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03 are 100nM.
(2) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension
It is adjusted to 30 × 104/ mL, the cell suspension inoculation of every hole 1.5ml is in 6 orifice plates.
(3) Lipofectamine is preparedTMThe compound of 2000 and RNA: according to LipofectamineTM2000 specifications,
The siRNA amount and Lipofectamine that each takes what he needsTM2000 amounts are diluted with Optimal Medium Opti-MEM respectively.
(4)LipofectamineTMAfter the completion of 2000 dilutions, it is stored at room temperature 5min.
(5) dilute and stand the Lipofectamine of 5minTM2000 with diluted SiRNA and mixed according to the ratio of 1:1,
It is stored at room temperature after 20min to get Lipofectamine is arrivedTM2000-RNA compound.
(6) by Lipofectamine needed for each groupTM2000-RNA compound is slowly added drop-wise to the cell suspension in orifice plate
In, so that the final volume in every hole is 2mL.6 orifice plates are placed in 37 DEG C, CO2In the cell incubator that volume fraction is 5%.
2.2.2 Real-time PCR detects the relative expression levels of H19mRNA in group of cells
2.2.2.1 Total RNAs extraction
Experimental group: blank control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03
(1) group of cells of logarithmic growth phase, 1500rpm are centrifuged 5min, abandon supernatant.
(2) PBS washes cell precipitation one time, and 1500rpm is centrifuged 5min, abandons supernatant.
(3) trizol of 1mL is added into cell precipitation, cracks 5min on ice.
(4) chloroform of 200 μ L is added into cell pyrolysis liquid, vibrates 45s, stands 10min on ice.
(5) 12000rpm, is centrifuged 15min by 4 DEG C.Careful supernatant of drawing is in a clean EP pipe, minus 20 DEG C of placements
20min。
(6) 12000rpm, is centrifuged 10min by 4 DEG C.Carefully discard supernatant.
(7) 70% alcohol washes RNA precipitate, and 12000rpm, is centrifuged 5min, in triplicate by 4 DEG C.
(8) precipitating is dried in draught cupboard, 2-3min.The RNA of 30 μ L is added in precipitating without enzyme water.RNA is placed in minus 80
DEG C save.
2.2.2.2 reverse transcription reaction (RT)
(1) PCR pipe for taking RNA sample 1 μ g of the A260/A280 between 1.8-2.0 and nuclease free to pollute, each group RNA
It is placed in spare on ice.
(2) according to the specification of the All-in-One cDNA Synthesis SuperMix of bimake company, in nucleic acid
Reverse transcription reaction (operating on ice) is carried out without following components in enzyme pipe, is added:
(3) after adding well according to said components, brief centrifugation 10s.
(4) PCR pipe is put into PCR instrument, the standard reaction program progress reverse transcription reaction provided according to kit: 42 DEG C
15min；85℃2min.
2.2.2.3 Real-time PCR
(1) each group is detected according to 2 × SYBR Green qPCR Master Mix kit of bimake company
The relative expression levels of H19mRNA, GAPDH are internal reference, and 1 μ L cDNA is taken to carry out Real-time PCR amplification.
(2) reaction system is as follows:
The relative expression levels of H19 and GAPDH are with 2-△△CTIt calculates.
Embodiment 3 establishes the K562 cell strain for stablizing expression H19
3.3.1 H19 Lentiviral and empty Lentiviral transfection K 562 cell
(1) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension
It is adjusted to 30 × 104/ mL, every hole 1.75mL cell suspension inoculation is in 6 orifice plates.
(2) Lipofectamine is preparedTM3000 in the compound of DNA: according to LipofectamineTM3000 specifications
It prepares, every hole takes the amount of DNA of 2.5 μ g and the Lipofectamine of 3.75 μ LTM3000 amounts, respectively with the Opti-MEM of 250 μ L
Dilution.
(3) DNA and LipofectamineTM3000 dilutions are completed, and dilution are uniformly mixed according to the ratio of 1:1, room temperature
After standing 5min, Lipofectamine is obtainedTM3000-DNA compound.
(4) by LipofectamineTM3000-DNA compound is slowly dropped in the cell suspension in orifice plate, and every hole is most
Final volume is 2mL.It is 37 DEG C, CO that six orifice plates, which are placed in temperature,2It cultivates in the cell incubator that volume fraction is 5%, after 6h, adds
Enter the RPMI-1640 culture medium that 2mL is 20% containing serum.
3.3.2 the K562 cell strain of expression H19 is stablized in puromycin screening
After H19 slow virus carrier transfection K 562 cell, puromycin is added in cultivating system, keeps the end of puromycin dense
Degree was 1mg/mL, every 2 days replacement culture solutions.
4 Real-time PCR of embodiment detects the relative expression levels of intracellular H19mRNA
4.4.1 Total RNAs extraction,
According to 2.2.2.1
4.4.2 reverse transcription reaction (RT)
According to 2.2.2.2
4.4.3 Real-time PCR
According to 2.2.2.3
The influence that 5 H19 of embodiment forms K562 cell colony
5.5.1 experimental group
1.K562 blank control group
2.K562-H19 LentiV group (the K562 cell for stablizing expression H19)
3.K562-empty LentiV group (containing the K562 cell for being free slow virus carrier)
5.5.2 COLONY is tested
Experiment is divided into two groups: stablizing the K562 cell of expression H19 and has transfected the K562 cell of empty Lentiviral.
The cell of each experimental group of logarithmic growth phase dilutes K562 cell with serum free medium, so that its density is 1 × 103/
ML takes the K562 cell suspension of 450 μ L, is added to 550 μ L containing 20% fetal calf serum, 5 μm of ol/L beta -mercaptoethanols, 2mmol/L
L-Glutamine and 0.9% methylcellulose semisolid culturemedium in, cell suspension and semisolid culturemedium is sufficiently mixed
After even, every hole 1mL is inoculated in 24 orifice plates, and each experiment repeats three multiple holes, and it is 37 DEG C, CO that 24 orifice plates, which are placed in temperature,2Body
It is cultivated 7-14 days in the cell incubator that fraction is 5%.It is placed under inverted microscope and observes COLONY size and form, with
The cell mass of approximately greater than 40 cells is 1 COLONY, and preservation of taking pictures, experiment is in triplicate.
Functional study of 6 H19 of embodiment in CML mouse model
6.6.1 the tumor formation of Balb/c nude mice by subcutaneous is tested
H19 slow virus carrier and empty slow virus carrier are transfected in P210 cell, and it is thin to construct the stable P210 for being overexpressed H19
Density is 1 × 10 by born of the same parents7200 μ L to the BALB/c nude mouse left lower extremity of P210-H19 and P210-NEG cell inoculation of/mL
Its tumor formation situation is observed in oxter, after mouse tumor formation, the daily variation for measuring mouse tumor size.
6.6.2 NOD/SCID mouse tail vein is tested
H19 slow virus carrier and empty slow virus carrier are transfected in K562 cell, and it is thin to construct the stable K562 for being overexpressed H19
Density is 1 × 10 by born of the same parents7The 200 μ L of K562-H19 and K562-NEG cell transplantation of/mL, tail vein injection K562-H19 cell
With K562-NEG cell, living imaging observes and records the morbidity and survival condition of mouse.
Experimental result:
Influence of the 1.H19 to K562 cell progression
The detection of 1.1 H19 expression quantity
The blood of normal person is taken, RNA is extracted, while to K562 cell, KCL-22 cell, 293T cell extraction RNA, fluorescence is fixed
The expression quantity for measuring PCR detection each group H19, as a result as shown in Fig. 2, compared with Normal group, the table of H19 in K562, KCL-22
It is lower up to measuring.
The screening of 1.2 H19-SiRNA ordered sequences
Concentration is SCR (serum creatinine), the H19-siRNA transfection K 562 cell of 100nm, after normal culture 48h, extracts each group
The total serum IgE of cell, Real-time PCR detect the relative expression levels of each group H19mRNA, MTT detection transfection H19-SiRNA
Afterwards, the cell viability of each group K562 cell.As a result as shown in Figure 3.
The Efficiency testing of 1.3 H19 slow virus carrier transfection K 562 cells
H19 slow virus carrier (CS-GS3160-LV201) used and empty slow virus carrier (CS-NEG-LV-201) be by
Provided by Guangzhou FulenGen Co., Ltd..H19 slow virus carrier screens after transfection K 562 cell through puromycin, obtains
To the K562 cell for stablizing expression H19.The each group K562 cell of logarithmic growth phase is collected, the phase of total serum IgE detection H19mRNA is extracted
To expression.The relative expression levels of the intracellular H19 of each group K562 are as shown in figure 4, relative to empty slow virus carrier (Empty
LentiV), the expression of the H19mRNA of H19 slow virus carrier group (H19LentiV) is significantly raised.
1.4 H19 are overexpressed the influence formed to K562 cell colony
The competence for added value that method can be used to detect single tumor cell is formed by the colony of carrier of methylcellulose.It takes
K562, K562-empty LentiV and K562-H19LentiV of logarithmic growth phase carry out colony experiment, as a result such as Fig. 5 institute
Show, relative to K562 group and K562-empty LentiV group, the colony number and size of K562-H19 LentiV group are reduced
(P<0.05)
2. being overexpressed influence of the H19 to CML model mouse Balb/c nude mice
Influence of the H19 overexpression to BALB/c nude mouse tumor size is probed into nude mice by subcutaneous tumor formation, to BALB/c
The influence of nude mouse survival, in order to probe into influence of the H19 to BALB/c nude mouse P210 tumor size, in P210 cell
Middle transfection H19 slow virus carrier and empty slow virus carrier, construct the P210 cell stablized and be overexpressed H19, are 1 × 10 by density7/
200 μ L to the BALB/c nude mouse left lower extremity oxter of P210-H19 and P210-NEG cell inoculation of mL, observes its tumor formation feelings
Condition, after mouse tumor formation, the daily variation for measuring mouse tumor size.As can be seen from Figure 6 the tumour ratio of H19 overexpression group
Control group it is small.
3. being overexpressed influence of the H19 to CML model mice NOD/SCID
Mouse tail vein injection K562-H19 cell and K562-empty cell, living imaging observe and record the morbidity of mouse
And survival condition.As a result as shown in Figure 7.
Interpretation of result:
The embodiment of the present invention is broadly divided into two parts, and function of the H19 in K562 cell is studied by first part.Second
It is studied the function of H19 in animal body is overexpressed part.
The expression quantity of H19 is detected first, the blood and K562 cell and KCL-22 cell of normal person is taken, extracts
RNA carries out H19 expression quantity and is detected.Such as Fig. 2, it can be seen that compared with Normal group, H19 in K562 and KCL-22
Expression quantity is relatively low.Secondly, having carried out the screening of ordered sequence to the SiRNA of H19, H19-siRNA and SCR synthesis are in Guangzhou
Sharp rich biological Co., Ltd.It being screened through Real-time PCR, H19-SiRNA-02 and H19-SiRNA-03 two sequences are effective,
It is unobvious to the effect of vigor of K562 cell such as Fig. 3, but after targeted inhibition H19.Therefore, inventor utilizes H19 slow virus carrier
Transfection K 562 cell, puromycin screens positive cell, so that H19 overexpression, stablizes strain and is named as K562-H19LentiV,
Research H19 is overexpressed the effect being in progress to K562 malignant, and such as Fig. 4, H19 are overexpressed the clone's shape for inhibiting K562 cell
At.Lentiviral CS-GS3160-LV201 and CS-NEG-LV-201 building and technical support are by Guangzhou reactivation gene public affairs
Department provides.CS-GS3160-LV201 contains H19 and eGFP target gene fragment, and CS-NEG-LV-201 contains only eGFP, institute
To observe the positive cell of GFP albumen after transfection is completed, transfection efficiency can be tentatively judged, it is anti-according to what is had on carrier
Property marker gene using puromycin carry out preliminary screening.Strain is stablized to the K562 after transfection slow virus and extracts RNA, Real-
The relative expression levels of time PCR detection H19.Fig. 5 shows compared with K562-empty LentiV groups of cells, K562-
The expression of H19LentiV intracellular H19mRNA is significantly raised, the above result shows that, stablize the K562 cell of expression H19
It is successfully established, this provides experimental model for function of the research H19 in CML.
Clonal hyperplasia is one important feature of CML K562 cell strain, therefore, to K562 cell clonal formation energy
Power, which carries out research, can prove influence of the H19 to CML cell progression.Colony forms experiment and refers to individual cells especially tumour
Upgrowth situation of the cell under methylcellulose semisolid culturemedium condition of culture, by inverted microscope observe cell number with
And colony form, it can be used to evaluate the proliferative capacity of individual cells.Experiment is formed by colony to learn, is overexpressed H19
The colony size and number of K562 cell are reduced afterwards, and the proliferative capacity of the expression and K562 cell of H19 is closely related.
Chronic myelocytic leukemia has characteristic BCR/ABL fusion and its product BCR/ABL fusion protein
(P210), the albumen have strong tyrosine kinase activity, can abnormal activation many A signal pathways, cause haemocyte pernicious
Conversion.BaF-P210 cell, abbreviation P210 are the cell strains for stablizing expression BCR/ABL.Therefore, this part is in animal water
The flat influence for having visited influence and survival that H19 is overexpressed to BALB/c nude mouse tumor formation.Inventor stablized constructing
Density is 1 × 10 by the P210 cell for expressing H197200 μ L to the BALB/c of P210-H19 and P210-NEG cell inoculation of/mL
From the mouse left lower extremity oxter nude is injected, the tumor formation situation and survival condition of mouse are observed.Fig. 6 can be seen that with it is right
It is compared according to group, the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.Meanwhile inventor is constructing
Stablize the K562 cell for being overexpressed H19, is 1 × 10 by density7K562-H19 the and K562-NEG cell of/mL is infused by tail vein
It penetrates, by 200 μ L to NOD/SCID Mice Body of cell transplantation, living imaging observes the incidence and survival condition of mouse,
From figure 7 it can be seen that compared with the control group, the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of virus expression carrier, which is characterized in that the carrier can express H19.
2. carrier according to claim 1, which is characterized in that the carrier can be overexpressed H19.
3. carrier according to claim 1, which is characterized in that the virus is slow virus.
4. carrier according to claim 1 or 3, which is characterized in that in the nucleic acid of the carrier containing CMV promoter and
H19 gene segment.
5. carrier according to claim 4, which is characterized in that also contain eGFP genetic fragment in the nucleic acid of the carrier.
6. a kind of cell strain, which is characterized in that the cell strain can stablize expression H19.
7. cell strain according to claim 6, which is characterized in that the cell strain is K562 cell strain.
8. cell strain according to claim 7, which is characterized in that contain the virus that can stablize expression H19 in the cell
Carrier.
9. cell strain according to claim 8, which is characterized in that the virus is slow virus, the nucleic acid of the slow virus
In contain CMV promoter, h19 gene segment and eGFP genetic fragment.
10. the carrier of Claims 1 to 5 and/or the cell strain of claim 6~9 are in the drug of preparation treatment malignant tumour
Application.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112494651A (en) * | 2020-09-30 | 2021-03-16 | 吉林大学 | Application of lncRNA H19 as molecular target in atherosclerosis treatment |
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2018
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| JING-DONG ZHOU等: "Hypomethylation-mediated H19 overexpression increases the risk of disease evolution through the association with BCR-ABL transcript in chronic myeloid leukemia", 《JOURNAL OF CELLULAR PHYSIOLOGY》 * |
| ZHENG Y，ET AL: "A comprehensive review of web-based non-coding ＲNA resources for cancer research", 《CANCER LETT》 * |
| 张婷娟等: "急、慢性髓系白血病患者中H19表达的临床意义及生物学功能研究", 《中国药理学会分析药理学专业委员会成立大会、第三届全国分析药理学学术大会暨贵州省药学会药学青年专业委员会成立大会论文集》 * |
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Cited By (1)
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| CN112494651A (en) * | 2020-09-30 | 2021-03-16 | 吉林大学 | Application of lncRNA H19 as molecular target in atherosclerosis treatment |
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Application publication date: 20181228 |