CN101954077B - Expression plasmid adjuvant for enhancing chemotherapeutic effect of tumor chemotherapeutics and preparation method thereof - Google Patents
Expression plasmid adjuvant for enhancing chemotherapeutic effect of tumor chemotherapeutics and preparation method thereof Download PDFInfo
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- CN101954077B CN101954077B CN 201010280657 CN201010280657A CN101954077B CN 101954077 B CN101954077 B CN 101954077B CN 201010280657 CN201010280657 CN 201010280657 CN 201010280657 A CN201010280657 A CN 201010280657A CN 101954077 B CN101954077 B CN 101954077B
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Abstract
The invention relates to the field of biomedicine. 5-fluorouracil (5-FU) is widely applied to the chemotherapy of gastral malignant tumors, but primary and acquired medicament resistant phenomena in the chemotherapy of colon cancer are universal; cell apoptosis is an important approach that chemotherapeutics play a role, and APAF-1 serving as regulatory protein is mainly involved in signal transduction of a mitochondrion approach of apoptosis; miR-23a can be combined with APAF1 serving as a target gene thereof so as to reduce the expression of the APAF1, and the apoptosis caused by the mitochondrion approach due to the chemotherapy is reduced so as to enhance the tolerance of the tumors to the chemotherapeutics; and the number of apoptotic cells is increased by reducing the expression amount of the in-vivo miR-23a so as to enhance the sensibility of the chemotherapeutics. The invention provides a PLKO-anti-miR-23a vaccine adjuvant for enhancing the chemotherapeutic effect of the chemotherapeutics, particularly the 5-FU. In-vitro experiments prove that the vaccine adjuvant can enhance the apoptosis level of colon cancer cells caused by the 5-FU serving as the chemotherapeutics obviously, and in-vivo experiments show that the vaccine adjuvant can enhance the chemotherapeutic effect of the 5-FU obviously, inhibit tumor growth and prolong the survival period of immunodeficient mice.
Description
Technical field
The present invention relates to biomedical sector, be specifically related to a kind of PLKO-anti-miR-23a expression plasmid adjuvant for strengthening the chemotherapeutics chemotherapy effect and preparation method thereof.
Background technology
5-fluorouracil (5-FU) is widely used in the chemotherapy of malignant tumor of digestive tract, due to its determined curative effect, is in other chemotherapeutic irreplaceable status always, but some cases are main chemotherapy and insensitive to this medicine.5-FU is the treatment colorectal cancer, gastric cancer, and the medicine that cancer of biliary duct is commonly used, the effective percentage of single therapy is 20% left and right only.Chemotherapeutic unite use (as: 5-FU+ vincristine+semustine), the chemotherapy effective percentage of advanced CRC is obviously improved, patient's median survival time obviously extends.However, colorectal cancer chemotherapy Central Plains is sent out with the acquired drug-resistance phenomenon very general.The gene genetic polymorphism makes the patient have individuation difference to chemosensitivity.The adjuvant of the chemotherapy regimen that How to choose is suitable and selective enhancement chemotherapy effect is benefited the patient from treatment, extremely important.
Apoptosis is the important channel that chemotherapeutics plays a role.Cell has propagation, differentiation and apoptosis three specific characters, and under normal circumstances, the three coordinates mutually, keeps balance.Tumor tissues is except having proliferation activity, and the apoptosis of tumor cells rate reduces, and the tumor cell Net growth rate will be improved, and prognosis is poorer.The process that occurs according to apoptosis is divided into two approach: one is death receptor pathway *, namely by the apoptosis enzyme caspase in the extracellular signal active cell; Article one, be mitochondria pathway, namely by the proteinase activated factor activator caspases of mitochondrion release apoptosis.The caspase albumen of these activation can with intracellular important protein degradation, cause apoptosis.Mitochondrion plays a crucial role in the apoptosis mechanism, it not only can self release cells pigment C etc. the material mediated apoptosis, can also and other apoptosis inducing factor combineds effect and change self active state, thereby better regulate apoptosis.APAF-1 (apoptosis peptide inducible factor 1) mainly participates in the signal transduction of mitochondria pathway as adjusting albumen.APAF-1 is distributed widely in the Various Tissues such as human brain, the heart, liver, spleen, lung, pancreas, kidney, thymus, small intestinal, colon, skeletal muscle, ovary, testis, peripheral blood leucocyte and cell.Under the chemotherapeutics effect, mitochondria dysfunction causes cytochrome C to discharge; Be discharged into cytoplasmic cytochrome C and can be combined with APAF-1 under the condition that dATP exists, make it form polymer, and impel caspase-9 with it in conjunction with formation apoptosis body, caspase-9 is activated immediately; The caspase-9 that is activated can activate other caspase, as caspase-3 etc.; Caspase-3 activates again DNA fragmentation factor, causes the endonuclease enzyme activation of quiescent condition, finally causes DNA break, causes apoptosis.
MicroRNA (miRNA) is the approximately non-coding protein strand microRNA of 21～25 bases of a class length, extensively be present in multicellular organism and virion, mainly mate by nucleic acid array complementation and be attached on specific said target mrna, suppressing said target mrna translation process or degraded said target mrna, is a kind of molecule that plays the negative regulation effect.More and more studies show that at present, the miRNA wide participation organism various physiological processes, and correlational study report shows that also its expression and functional disorder may affect the multiple pathological phenomenons such as tumorigenesis and chemotherapy.At present, the report for miRNA intervention chemotherapy of tumors increases gradually.Such as: the people such as FANYIN MENG confirm: the expression that suppresses miR-21 and miR-200b in the chemotherapy of cancer of biliary duct can strengthen sensitivity (the FANYIN MENG of chemotherapeutics, ROGERHENSON, MOLLY LANG, HANIA WEHBE, SHAILMAHESHWARI, JOSHUA T.MENDELL, JINMAI JIANG, THOMAS D.SCHMITTGEN, and TUSHAR PATEL GASTROENTEROLOGY2006; 130:2113-2129).Chan JA, Krichevsky AM, find that Deng researcher miR-21 can strengthen by the expression of disturbing apoptosis-related genes gliomatous drug resistance (Chan JA, Krichevsky AM, Kosik KS.MicroRNA-21is an antiapoptotic factor in humanglioblastoma cells.Cancer Res.2005Jul 15; 65 (14): 6029-33.).
Hsa-mir-23a (Mature sequence MIMAT0000078) be a kind of after the tumors such as human bile duct cancer, gastric cancer, colon cancer and breast carcinoma are processed through chemotherapeutics 5-FU the miRNA of high expressed.Present report for hsa-mir-23a, for example: in cancer of biliary duct, the expression of mir-23a is significantly raised, (FANYIN MENG, ROGER HENSON, MOLLY LANG, HANIAWEHBE, SHAIL MAHESHWARI, JOSHUA T.MENDELL, JINMAI JIANG, THOMAS D.SCHMITTGEN, and TUSHAR PATEL GASTROENTEROLOGY2006; 130:2113-2129); After colon cancer is processed through the 5-FU chemotherapy, the expression of miR-23a obviously raises, (Lorena Rossi, Enzo Bonmassar, Isabella Faraoni, Modification of miRgene expression pattern in human colon cancer cells following exposure to5-fluorouracil in vitro Pharmacological Research 56 (2007) 248-253); MiR-23a strengthens gliomatous formation by the adjusting of expressing for LMNB1 and myelin forms, thereby promote propagation (the Shu-Ting Lin of neuroglial cytoma, Ying-Hui Fu, miR-23regulation oflamin B1 is crucial foroligodendrocyte development and myelination DiseaseModels ﹠amp; Mechanisms 2,178-188 (2009) doi:10.1242/dmm.001065).
The research of the expression plasmid adjuvant of so far there are no a kind of expression miRNA-23a inhibitor for strengthening the chemotherapy of tumors effect and should being used for strengthens the report of the chemotherapy effect of tumor.
Summary of the invention
The object of the present invention is to provide a kind of expression plasmid adjuvant for strengthening the tumor chemotherapeutic drug chemotherapy effect and preparation method thereof.
The present invention's imagination, miR-23a can be combined with its target gene APAF1, thereby reduces the expression of APAF1 gene, reduces the apoptosis of the mitochondria pathway that causes because of chemotherapy.Thereby strengthen tumor to the toleration of chemotherapeutics.On the contrary, cross the inhibitor of expressing miR-23a, make the expression decline of miR-23a in body can cause that apoptosis cell increases, thereby strengthen the sensitivity of chemotherapeutics 5-FU.Therefore the present invention's inhibitor expression plasmid of intending in vivo or directly changing in tumor miR-23a can strengthen the chemosensitivity of tumor.
The invention provides a kind of expression plasmid adjuvant for strengthening the tumor chemotherapeutic drug chemotherapy effect, it contains the positive-sense strand of neck ring structure take slow virus carrier plasmid PLKO.1 as basic framework: 5 ' CCGGTATCACATTGCCAGGGATTTCCCTCGAGGGAAATCCCTGGCAATGTGATTTT TTG 3 ' and antisense strand: the plasmid of 5 ' AATTCAAAAAATCACATTGCCAGGGATTTCCCTCGAGGGAAATCCCTGGCAATGTG AT A, 3 ' sequence.
But the expression plasmid that the ripe body sequence of above-mentioned plasmid stably express and miR-23a (AUCACAUUGCCAGGGAUUUCC) is complementary.The expressed sequence of this expression plasmid is cloned in other expression vector and expresses the expression plasmid adjuvant that also can build other.
Above-mentioned tumor chemotherapeutic drug is 5-fluorouracil (5-FU) or can causes the chemotherapeutics that miR-23a expresses to be increased in chemotherapy of tumors.
When expression plasmid adjuvant of the present invention and 5-FU use in conjunction, can improve tumor for the sensitivity of 5-FU, improve the chemotherapy effect of tumor.This vaccine can oneself cause that apoptosis increases simultaneously, therefore also can should be used for strengthening chemotherapy effect with other chemotherapy drugs in combination.This vaccine can be used as the expression plasmid adjuvant that strengthens chemotherapy effect and uses.
The invention provides a kind of expression plasmid adjuvant for strengthening the tumor chemotherapeutic drug chemotherapy effect; by this plasmid of direct injection in tumor or by the implant region slow-releasing pump, plasmid to be injected in the tumor body; allow this adjuvant constantly increase in the tumor body and express the inhibitor of miR-23a; thereby inhibitor is combined with the miR-23a of inside tumor; suppress the pressure of tumor endogenous cause of ill chemotherapeutics and cause excessively expressing of miR-23a; thereby be to strengthen when chemotherapy drugs in combination is used the sensitivity of tumour medicine, strengthen chemotherapy effect.The application mode of plasmid adjuvant of the present invention comprises: intratumor injection, intramuscular injection, subcutaneous injection, intravenous injection, lumbar injection, mucosa absorption, the application modes such as gastrointestinal absorption.Can use with chemotherapy drugs in combination also can be at the tumor by local intratumor injection.
The present invention also provides a kind of preparation method of the plasmid adjuvant for strengthening the tumor chemotherapeutic drug chemotherapy effect, and concrete steps are as follows:
Expression miR-23a inhibitor sequence with neck ring structure:
Positive-sense strand: 5 ' CCGGTATCACATTGCCAGGGATTTCCCTCGAGGGAAATCCCTGGCAATGTGATTTT TTG 3 ' (SEQ ID NO:1)
Antisense strand: 5 ' AATTCAAAAAATCACATTGCCAGGGATTTCCCTCGAGGGAAATCCCTGGCAATGTG AT A 3 ' (SEQ ID NO:2)
Add at the two ends of sequence and can be combined with the carrier terminal matching sticky end of restriction enzyme site, will with the sequence of neck ring structure after synthetic, nucleotide sequence be resuspended in distilled water, concentration is 1ug/ul.With the synthetic duplex of this tube nucleus nucleotide sequence annealing.
The annealing system is:
-5ul?of?Sense?oligo
-5ul?of?Antisense?oligo
-5ul?of?10x?NEB?buffer?2
-35ul?ddH2O
Altogether the 50ul system, hatched 4 minutes in 95 ℃ of water-baths, hatched 10 minutes in 70 ℃ in the beaker that fills 1500ml water, then naturally is down to room temperature, the formation duplex structure.Slow virus carrier plasmid PLKO.1 after restriction enzyme site AgeI and EcoRI enzyme action, is entered the double chain oligonucleotide sequence clone in PLKO.1 carrier after enzyme action.Be transformed in the escherichia coli competence, aim sequence in a large number increases.After identifying correctly, order-checking carries out next step applying detection.
The present invention shows through results of animal: 1. after passing through transfection expression in the in-vitro transfection CCL188, further identify that by realtime PCR the expression of miR-23a changes situation, determine that further the miRNA-23a inhibitor sequence of this plasmid expression is at external stably express (as shown in Figure 3).2. inoculate transplanted tumor in Immune deficient mice, by this adjuvant of direct intratumor injection or by setting up the colon cancer cell line that surely turns PLKO-anti-miR23a, and be seeded in the impact of further observing in Immune deficient mice the change of chemotherapy effect.This vaccine adjuvant of proving again is stably express in vivo, and the chemotherapy of tumor has been played potentiation.Therefore proved that PLKO-anti-miR23a can be used as vaccine adjuvant and bring into play obvious antitumor action in chemotherapy of tumors.3. provided by the invention the amplification in a large number by escherichia coli expressed the plasmid of miR23a inhibitor, and the method for the plasmid amplification in vitro of maturation has ripe Amplification Technologies and isolation and purification method at present.4. with the plasmid of escherichia coli amplification in vitro miR23a inhibitor, produce simply, with low cost, give the interior injection operation of patient body convenient, can repeatedly inject plasmid after the implantation slow release pump, little to patient trauma.5. compare with other tumor chemotherapeutic drugs, directly the intratumor injection side effect is little.
Description of drawings
Fig. 1: be PLKO-anti-miR23a expression plasmid restructuring strategic map;
Fig. 2: be recombiant plasmid sequencing result figure.
Fig. 3: the in-vitro transfection recombiant plasmid identifies that by realtime PCR miR-23a expresses the cartogram that changes.
The specific embodiment:
The invention will be further described below in conjunction with drawings and Examples, but enforcement of the present invention is not limited only to this.
Embodiment 1: the structure of recombiant plasmid PKLO-anti-miR23a
Design has the expression miR-23a inhibitor sequence of neck ring structure:
Positive-sense strand: 5 ' CCGGTATCACATTGCCAGGGATTTCCCTCGAGGGAAATCCCTGGCAATGTGATTTT TTG 3 '
Antisense strand: 5 ' AATTCAAAAAATCACATTGCCAGGGATTTCCCTCGAGGGAAATCCCTGGCAATGTG AT A 3 '
Add at the two ends of sequence and can be combined with the carrier terminal matching sticky end of restriction enzyme site, will with the sequence of neck ring structure after synthetic, nucleotide sequence be resuspended in distilled water, concentration is 1ug/ul.With the synthetic duplex of this tube nucleus nucleotide sequence annealing.
The annealing system is:
-5ul?of?Sense?oligo
-5ul?of?Antisense?oligo
-5ul?of?10x?NEB?buffer?2
-35ul?ddH2O
Altogether the 50ul system, hatched 4 minutes in 95 ℃ of water-baths, hatched 10 minutes in 70 ℃ in the beaker that fills 1500ml water, then naturally is down to room temperature, the formation duplex structure.Slow virus carrier plasmid PLKO.1 (Openbiosystems USA) after restriction enzyme site AgeI and EcoRI enzyme action, is entered the double chain oligonucleotide sequence clone in PLKO.1 carrier after enzyme action (as shown in Figure 1).Be transformed in the escherichia coli competence, choose the monoclonal aim sequence that further increases.After identifying correctly, the order-checking of trust Invitrogen company carries out next step applying detection (as shown in Figure 2).
Embodiment 2: a large amount of preparations of recombiant plasmid PKLO-anti-miR23a (adopting the plasmid of green skies biotech company to take out greatly test kit)
1. get in bacterium to the 50 milliliter centrifuge tube that spends the night, 5000g collected bacterial precipitation in centrifugal 1 minute, abandoned supernatant.Repeat once, every pipe is collected 100 milliliters of bacterium precipitations of spending the night altogether again.
2. every pipe adds 5 ml soln I, resuspended bacterial precipitation.Guarantee to precipitate and scatter fully, without visible bacterial aggregate.Confirm to have added RNase A in solution I.Maximum speed vortex 10-20 second or longer time, hanged precipitation.Fully mixing, should be uniform suspension facing to light place's observation, and nothing is bacterial aggregate or wadding piece obviously.If there is no the vortex instrument, can precipitation be scattered gradually with rifle piping and druming precipitation.
3. every pipe adds 5 ml soln II, puts upside down centrifuge tube 4-6 time gently, and room temperature was placed 3-5 minute, made the complete cracking of antibacterial, and solution is transparent.
4. every pipe adds 5 ml soln III, puts upside down immediately 4-6 mixing of centrifuge tube, and visible white floccule produces.
5. the most at a high speed (＞5,000rpm) the centrifugal 10-20 of room temperature minute.If speed is higher by for example 10, the 000rpm left and right, general centrifugal 10 minutes are enough.Can be ready to plasmid purification column when centrifugal, self-control funnel etc., and put on mark on purification column.
6. the supernatant after previous step is centrifugal is poured into or is drawn in plasmid purification column.High speed centrifugation 2 minutes is abandoned the collection liquid in pipe.After plasmid is poured plasmid purification column into, can wait for, directly centrifugal.After abandoning the liquid in collecting pipe, keep collecting pipe and continue to use.
7. add 7 ml soln IV in plasmid purification column, high speed centrifugation 2 minutes washes away impurity, abandons the collection liquid in pipe.Can wait for after adding solution IV, directly centrifugal.After abandoning the liquid in collecting pipe, keep collecting pipe and continue to use.
8. the most centrifugal 2 minutes, remove residual liquid and trace ethanol is volatilized fully.Attention: abandon and collect after liquid in pipe centrifugally again, could thoroughly remove the solution IV of trace.The solution IV of trace can affect the quality of plasmid.
9. plasmid purification column is placed on 50 milliliters of centrifuge tubes, adds 2 ml soln V to managing on inner cylinder, placed 2 minutes.Also can be with redistilled water or MiliQ level pure water replace solution V, but the pH of water should be not less than 6.5.Solution V adds rear standing time slightly long, can be slightly helpful for increasing yield plasmid.10. high speed centrifugation is 2 minutes, adds 10 ml soln VI in gained liquid, is added to after mixing in former plasmid purification column.Adding must mixing after solution VI! Can put upside down mixing, be sure not centrifugal after mixing.
11. high speed centrifugation 2 minutes is abandoned the collection liquid in pipe.
12. add 15 ml soln IV in plasmid purification column, high speed centrifugation 2 minutes further washes away impurity, abandons the collection liquid in pipe.Can wait for after adding solution IV, directly centrifugal.After abandoning the liquid in collecting pipe, keep collecting pipe and continue to use.
13. the most centrifugal 2 minutes, remove residual liquid and trace ethanol volatilized fully.Attention: abandon and collect after liquid in pipe centrifugally again, could thoroughly remove the solution IV of trace.The solution IV of trace can affect the quality of plasmid.
14. plasmid purification column is placed on 50 milliliters of centrifuge tubes, add 2 ml soln V to managing on inner cylinder, placed 2 minutes.
15. high speed centrifugation 2 minutes, gained liquid is ultrapure plasmid.
16.DNA quantitative analysis is with the TE buffer dilution DNA solution of appropriate volume.With ultraviolet spectrophotometer measuring device OD
260And OD
280, the content of calculating DNA.OD
260/ OD
280The content 300ng/ul of=1.82DNA.
Embodiment 3:PLKO-anti-miR23a strengthens chemotherapeutic 5-FU to the chemotherapy effect of colon cancer transplanted tumor
One, the foundation of human colon cancer cell strain HCT116 transplanted tumor model:
To collect 1 * 10
7Individual HCT116 cell (available from Chinese Academy of Sciences's cell bank) is resuspended with serum-free medium, is inoculated in back, 6-8 week Immune deficient mice right side subcutaneous, inoculates that after 10 days, tumor grows, and treats that Mice Body is made as 100mm
3The time, mice is divided into three groups at random.A: tail vein injection 60 a μ l/ PBS; B: tail vein injection 60 μ l/ 5-FU+ intratumor injection PLKO.1 empty plasmids; C: tail vein injection 60 μ l/ 5-FU+ intratumor injection PLKO-anti-miR23a expression plasmids.
Measure weekly the tumor length and width, application of formula V=0.4 * LW
2Calculate gross tumor volume, tail vein injection plasmid and chemotherapeutics 5-FU are once in a week.
Result shows, intratumor injection PLKO-anti-miR23a expression plasmid+5-FU tail vein injection group obviously suppresses tumor growth, and its successful that suppresses tumor growth is stronger than using separately 5-FU tail vein injection group.
Two, the foundation of human colon cancer cell strain HT29 transplanted tumor model
To collect 1 * 10
7Individual HT29 cell (available from Chinese Academy of Sciences's cell bank) is resuspended with serum-free medium, is inoculated in back, 6-8 week Immune deficient mice right side subcutaneous, inoculates that after 10 days, tumor grows, and treats that Mice Body is made as 100mm
3The time, mice is divided into three groups at random.A: tail vein injection 60 a μ l/ PBS; B: tail vein injection 60 μ l/ 5-FU+ intratumor injection PLKO.1 empty plasmids; C: tail vein injection 60 μ l/ 5-FU+ intratumor injection PLKO-anti-miR23a expression plasmids.
Measure weekly the tumor length and width, application of formula V=0.4 * LW
2Calculate gross tumor volume, tail vein injection plasmid and chemotherapeutics 5-FU are once in a week.
Result shows, intratumor injection PLKO-anti-miR23a expression plasmid+5-FU tail vein injection group obviously suppresses tumor growth, and its successful that suppresses tumor growth is carefully stronger than using separately 5-FU tail vein injection.
Claims (1)
1. plasmid adjuvant that be used for to strengthen the tumor chemotherapeutic drug chemotherapy effect, it contains the plasmid of the positive-sense strand sequence as shown in SEQ ID NO:1 and the antisense strand sequence as shown in SEQ ID NO:2 of neck ring structure take slow virus carrier plasmid PLKO.1 as basic framework; Wherein said tumor chemotherapeutic drug is 5-fluorouracil; Wherein said chemotherapy effect is the chemotherapy effect of colon cancer; And be with slow virus carrier plasmid PLKO.1 after restriction enzyme site AgeI and EcoRI enzyme action, the double chain oligonucleotide sequence clone is entered in PLKO.1 carrier after enzyme action.
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| CN108456670B (en) * | 2017-02-21 | 2021-05-18 | 中国科学院生物物理研究所 | Use of a magnetic field confining device for the preparation of a product for assisting chemotherapy |
| CN112494654B (en) * | 2020-12-10 | 2021-12-17 | 暨南大学附属第一医院（广州华侨医院） | Pharmaceutical composition containing LncRNA HCG18 inhibitor and application thereof |
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