CN1160130C - Plasmid used directly in intramuscular injection for treating diabetes - Google Patents

Abstract

The present invention provides plasmids for treating diabetes by direct intramuscular injection. The plasmids are carriers pcDNA3.1(-) which contain CGRP genes (CGRP-human calcitonin gene related peptide), wherein the CGRP genes can be expressed under the guidance of CMV promoters in pc DNA 3.1 (-).

Description

A kind of plasmid that is used for direct intramuscular injection for treating diabetes

Technical field

The present invention relates to a kind of plasmid that is used for direct intramuscular injection for treating diabetes.The eukaryotic expression recombinant plasmid that the present invention will contain hCGRP (CGRP) gene directly imports skeletal muscle tissue, makes it to express CGRP albumen, realizes immunomodulating, so that type 1 diabetes is carried out gene therapy.

Technical background

Gene therapy is to combine with molecular biology along with modern medicine over past ten years and be born, and it is meant the transfer by gene, and genes of interest is imported in patient's body, makes it to remedy the sudden change of original gene or express defective, reaches the purpose of treatment.Genes of interest is meant the exogenous normal gene that remedies functional impairment.The gene transfer approach can divide two classes: a class is the genes of interest direct injection, is about to have the virus of genes of interest, the liposome that is enclosed with genes of interest or exposed genes of interest and is injected directly in the test individuality; Another kind of is that the cell of cell line or subjects is taken out, In vitro culture, and import genes of interest with modes such as virus, liposomees, then the cell of these genetic modifications is failed back again and tested in the individual body.The direct route of metastasis of genes of interest is easy and simple to handle, promote easily, but these class methods immaturity still at present exists that curative effect is short, problems such as immunologic rejection and safety.The cell transfer method is relatively more classical, and effect is more easy to control, but it exists step many, and the technical sophistication difficulty is big, the problem that is not easy to implement.The importing of genes of interest can be made carrier with virus or plasmid.Though viral vector has the high advantage of transfection efficiency, has safety issue.Retrovirus DNA may insert in the host cell gene group at random, causes cancerating of cell; But the immunoreation of the expression product excitating organism of adenovirus itself.With the plasmid be carrier separately or wrap up direct injection with liposome etc. and shift, show it is feasible through many experiments, genes of interest has obtained expression, and the structure of plasmid, a large amount of preparations also are easier to, its genetic fragment of carrying is also relatively large.Liposome can improve plasmid DNA to intracellular transfer, and electroporation can improve the conversion ratio of plasmid DNA equally, but report genes of interest high level expression is arranged more than 6 months.It is a lot of that genes of interest injects intravital position, can decide according to therapeutic purposes, and there are skeletal muscle, cardiac muscle, liver, spleen, intracranial, peritoneum, subcutaneous tissue, vein, arterial wall etc. in the position of existing report.Skeletal muscle is big because of its capacity, cell survival long, synthesis secretion enlivens reasons such as reaching processing ease, is most widely used.

Type 1 diabetes be the result that genetic predisposition and environmental factors combined effect cause autoimmune damage.Because the allelic existence of diabetes susceptibility or lack corresponding protection gene (infer majority may for regulating immunoreactive gene), make that the autoimmune response at the B cell is easy to take place.Immune regulation mechanism unbalance mainly is the forfeiture of T cellular immunization balance stable state, starts autoimmune response, and then infringement of carrying out property B cell and afunction, the insulin secretion minimizing, and blood sugar increasing finally shows as clinical diabetes.

In the autoimmune pathogenesis of diabetes, T lymphocyte, especially CD4 ⁺The auxiliary lymphocyte of T played the part of important role.CD4 ⁺The T cell can be divided into Th1 subgroup (secretion IL-2, IFN-γ etc.) and Th2 subgroup (secretion IL-4, IL-10 etc.) by its excretory cytokine difference on function.The naive T cell that produces in the thymus (naive T lymphocytes) is subjected to dissimilar antigenic stimulus, under the cytokine and immunocyte effect of complexity, is divided into Th1, Th2 subgroup.Two big Th subgroups can keep relative subgroup stability, antagonism, inhibition mutually mutually in immune response.The excretory inflammatory cytokine that causes of Th1 cell subset can promote the autoimmune inflammation infringement; The next effect of the excretory cytokine ordinary circumstance of Th2 cell subset with antagonism autoimmune inflammation.Human body and zooperal a large amount of evidence show that the Th1/Th2 subgroup is unbalance to be the major reason that causes the B cell infringement.At the unbalance experimental treatment of carrying out immunologic intervention of this T cell function, might obtain to prevent and treat the effective means of such disease from cause of disease level.

CGRP is the neuropeptide with important immunoregulation effect.Mainly be present in people and the mammiferous sensory nerve.Existing result of study shows, CGRP can be under the situation of exsomatizing, suppress the secretion of the lymphocytic conversion of T and IL-2 and IFN-γ, thereby changed the contrast of Th1/Th2 subgroup, and then the morbidity and the state of an illness of autoimmunity pathological changes such as type 1 diabetes produced preventive and therapeutic effect.

Summary of the invention

The purpose of this invention is to provide a kind of plasmid that is used for direct intramuscular injection for treating diabetes.

We have studied CGRP adjusting to the Th cell function under the body situation emphatically.CGRP is as peptide class neurotransmitter, and the half-life only has several minutes, and the polypeptide high price of synthetic, carries out the immunomodulating intervention with the CGRP direct injection, and difficulty is very big.We have made up the recombiant plasmid that contains human CGRP's gene, founded CGRP gymnoplasm grain intramuscular injection gene transfer method, have obtained steady in a long-term expressing at body CGRP easily, have observed under the body situation CGRP to the regulating action of Th cell subsets.We have obtained the splenic T lymphocyte of the autoimmune diabetes mice of CGRP therapeutic transgene, its breeder reaction and cytokine secretion have been measured, find that the CGRP therapeutic transgene has suppressed ConA activated T cell proliferation, and having suppressed the secretion of Th1 cytokine IFN-γ, IL-4 does not have inhibitory action to the Th2 factor.Also significantly reduce by the excretory IL-12 of macrophage in the therapeutic transgene pneumoretroperitoneum liquid, may suppress the Th1 subgroup from level of differentiation.This selectivity suppresses to change the ratio of IFN-γ/IL-4, has regulated the balance of Th1/Th2 subgroup in the body.T cellular immune mechanism from the autoimmune diabetes morbidity, we confirm that the CGRP of transgene expression is by suppressing the selectivity that causes inflammatory Th1 subgroup function, alleviated the infringement of insulitis and B cell, and finally reduced the sickness rate of Autoimmune Diabetes, alleviated the diabetes state of an illness.

The invention provides a kind of plasmid that is used for direct intramuscular injection for treating type 1 diabetes, this plasmid is the carrier pcDNA3.1 (-) that contains the CGRP gene, and wherein, the CGRP gene can be expressed under the guiding of the CMV promoter among the pcDNA3.1 (-).CGRP gene of the present invention contains the nucleotide sequence of the 2nd, 3,5,6 exons of people CT/CGRP gene.

Brief Description Of Drawings

Fig. 1 eukaryon expression plasmid pcDNA3.1 (-) collection of illustrative plates.

The CMV-cytomegalovirus; The PolyA-polyadenylic acid; EcoRI, BamHI, HindIII-restricted enzyme; T7-promoter code name; BGHpA-bovine growth hormone polyadenylic acid; Ampicilin-ampicillin gene; ColE1-plasmid replication starting point; SV40 ori-simian virus replication origin; SV40pA-simian virus polyadenylic acid; Fl ori-filobactivirus replication origin; The kb-kilobase is right; The Neomycin-neomycin; The ori-replication origin; The pCMV-cytomegalovirus promoter; Fragment between the restriction endonuclease NheI PmeI is the multiple clone site of pcDNA (-), and target DNA is connected between the segmental restriction endonuclease EcoRI/HindII of this polyclone and between the EcoRI/BamHI.

Fig. 2 CGRP construction of recombinant plasmid sketch map.

The CGRP gene is cloned among the eukaryon expression plasmid pcDNA3.1 (-) and is subjected to the control of promoter CMV.

The variation of Fig. 3 multiple low dose STZ injection back mouse blood sugar and insulin.

Every day lumbar injection 40mg/kg dosage streptozotocin (STZ), injected continuously 5 days, get 1 from the tail vein of mice and bleed respectively at the different time after the injection, measure blood sugar concentration with Glucotrend blood sugar detection instrument, observe blood sugar concentration and change; Get the about 0.5mL of blood from eyeball of mouse, centrifugal 10 minutes of 4000g measures the mice serum insulin concentration with insulin human multi-resistance antibody and radioimmunoassay method, and (Fig. 3 A is a blood glucose, and B is an insulin to observe insulin level.n＝5； ^*：p＜0.05)

CGRP protein content increase in skeletal muscle tissue and the blood plasma after the intramuscular injection of Fig. 4 CGRP plasmid.

CGRP is measured by radioimmunoassay in plasmid injection back skeletal muscle homogenate and the blood plasma, and Fig. 4 A is skeletal muscle homogenate, and B is a blood plasma.As seen behind the therapeutic transgene, all than obviously increasing before the transgenic, the amplitude of increasing is 1-3 times to the content of CGRP, more than expression time in sustainable 4 weeks in mice plasma and the skeletal muscle.(n≥6； ^*：p＜0.05)

The expression of skeletal muscle CGRP mRNA after the intramuscular injection of Fig. 5 RT-PCR method mensuration CGRP plasmid.

Respectively at the skeletal muscle that extracted the plasmid injection areas behind the transgenic on the the 3rd, 14,28 day, measure the level of its CGRP mRNA with the RT-PCR method, discovery normal control C57BL mouse, injection empty plasmid control mice all do not have CGRP mRNA and express, and in the skeletal muscle of CGRP transgenic mice, at transgenic the expression of CGRP mRNA and sustainable reaching more than 4 weeks were arranged in the time of 3 days.RT-PCR electrophoresis photo: 1 normal C57BL mouse, 2pcDNA3.1 empty plasmid transgenic, 3CGRP plasmid transgenic 3 days, 4CGRP plasmid transgenic 14 days, 5CGRP plasmid transgenic 28 days, the negative control of 6 no amplification templates, the positive control of 7CGRP plasmid, MMarker.

The injection of Fig. 6 CGRP plasmid is to the influence of autoimmune diabetes mouse T cell propagation.

Use ³H-TdR mixes method, has compared empty plasmid matched group and the CGRP transgenic group diabetic mice splenic T lymphocyte multiplication effect under external antigens c onA stimulates, result such as Fig. 6.The T lymphocyte of CGRP therapeutic transgene diabetic mice is under ConA (2 μ g/ml) stimulates, and its multiplication effect significantly is lower than empty plasmid injection matched group (n=5; ^*: p＜0.05).

Fig. 7 CGRP transgenic is to the excretory influence of autoimmune diabetes mouse spleen T lymphocyte factor.

By measuring its functional status of the excretory cytokine analysis of activated T lymphocytes, wherein IFN-γ reflection causes the function of inflammatory Th1 subgroup, and IL-4 then reflects Th2 subgroup function.Fig. 7 A and B are presented at ConA (2 μ g/ml) or anti-mice CD3 antibody/PMA (0.005 μ g/ml) stimulates down, the difference of empty plasmid matched group and CGRP therapeutic transgene group mouse T cell IFN-γ and IL-4 secretory volume.As seen the CGRP therapeutic transgene has significantly reduced the secretion that causes inflammatory Th1 factor IFN-γ, but the secretion that presses down inflammatory Th2 factor IL-4 is not had influence.The T cell is taken from the mice that STZ injects back 7 days (behind the CGRP transgenic 12 days). ^*：p＜0.05，n＝4。

Fig. 8 CGRP transgenic is to the influence of mice Autoimmune Diabetes sickness rate.

The sickness rate of paired observation empty plasmid matched group and CGRP plasmid injection transgenic group mice diabetes, being higher than 12mmol/L with blood glucose is the onset diabetes standard.As seen the CGRP transgenic has significantly reduced the sickness rate that STZ causes Autoimmune Diabetes, reduces to 23% by 73% during 4 weeks.CGRP therapeutic transgene group and each 30 mice of empty plasmid injection matched group are compared two groups of sickness rate with the x2 check, ^*: p＜0.05.

Fig. 9 CGRP transgenic is to the influence of blood glucose in diabetic mice and plasma insulin level.

Compare the average blood sugar level of CGRP therapeutic transgene group and empty plasmid matched group, CGRP plasmid injection group significantly reduces, and continues more than 4 weeks, and blood glucose reduction amplitude reaches 3mmol/L (Fig. 9 A) when 4 weeks.Each 30 mice of CGRP therapeutic transgene group and empty plasmid injection matched group are with the MBG relatively of t check between two groups. ^*：p＜0.05。

Adopt the measured by radioimmunoassay plasma insulin level, consistent with the result that blood glucose improves, CGRP treatment group insulin level significantly increases (Fig. 9 B) than the empty plasmid matched group.Two groups each get 7 mices at random and get the hematometry insulin, with the insulin mean relatively of t check between two groups. ^*：p＜0.05。

Embodiment

1 contains the structure of the eukaryotic expression recombinant plasmid of human CGRP's gene

The eukaryon expression plasmid pcDNA3.1 (-) that this experiment is used (Fig. 1) is Invitrogen company (Invitrogen Corporation, 1600 Faraday Ave.PO Box 6482, Carlsbad, CA92008, USA) commercial goods.Through enzyme action, the cDNA segment that contains the CGRP gene that reclaims 844bp obtains human CGRP's gene by the PTT42 plasmid.Connect the downstream of the CMV promoter in plasmid pcDNA3.1 (-), and be subjected to the control (the CGRP gene order is seen SEQ IDNO:1) of CMV promoter.The construction of recombinant plasmid process is seen Fig. 2.

2 diabetic mice Preparation of model

Adopt classical way, select body weight 18-20 gram, the male C 57 BL mouse in 6-8 week is injected the streptozotocin (STZ) of 40mg/kg dosage every day, injects continuously 5 days, observes blood glucose and insulin concentration and changes (Fig. 3 A and B).Mice is put to death in 7 days, 2 weeks, 4 weeks, 8 weeks behind injection STZ, gets the capable histological examination of pancreas and confirms that islets of langerhans has the volume lymphocytic infiltration, and the islets of langerhans atrophy is the autoimmunity insulitis of cell mediated, meets the tissue performance of type 1 diabetes.

The variation of CGRP in skeletal muscle and the blood plasma after the intramuscular injection of 3CGRP plasmid

Gave therapeutic transgene the same day before the STZ injection first time.Use 100 μ l glass microsyringes to draw CGRP plasmid and contrast pcDNA3.1 empty plasmid (all being diluted to 1 μ g/ μ l), with 100 μ g/ mouse muscle injections with physiological saline solution.The injection site is the hind leg gastrocnemius, each 50ug of every side, and after the injection, the electric current with 100v, 40ms, 10 pulses, 1Hz carries out electricity irritation at once.CGRP content is measured by radioimmunoassay in plasmid injection back skeletal muscle homogenate and the blood plasma, sees Fig. 4 A and B.As seen behind the therapeutic transgene, the content of CGRP all obviously increases than control mice in mice plasma and the skeletal muscle, and the amplitude of increasing is 1-3 times, more than expression time in sustainable 4 weeks.

The expression of 4 plasmids injection back CGRP gene mRNA in the mice skeletal tissue

To cutting the about 0.5g of mice tibialis anterior injection site skeletal muscle tissue on the 3rd, 14,28 day behind the diabetic mice injection CGRP plasmid, extract total RNA, with RT-PCR method detection foreign DNA transcription product mRNA in muscular tissue.Mice with injection pcDNA3.1 (-) is contrast.MRNA with β-action is the contrasts of PT-PCR confidential reference items.The result shows that normal control C57BL mouse, injection empty plasmid control mice all do not have CGRP mRNA and express, and in the skeletal muscle of CGRP transgenic mice, at transgenic the expression of CGRP mRNA is arranged in the time of 3 days, and sustainablely reaches 4 weeks above (Fig. 5).The method that this has proved the muscle direct injection that the present invention uses can make external source human CGRP gene express in skeletal muscle.

The injection of 5CGRP plasmid is to the influence of T lymphocyte immunologic function and autoimmune response

Be the CGRP of the expression of recombinant plasmid that observe the to import effect to the mouse T cell immunity, our separation and Culture splenic T lymphocyte is measured the change of T cell proliferation and cytokine secretion behind the CGRP transgenic.The multiplication effect of transgenic group diabetic mice splenic T lymphocyte under external antigens c onA stimulates significantly is lower than empty plasmid contrast (Fig. 6); And the CGRP therapeutic transgene has also significantly reduced the secretion that causes inflammatory Th1 factor IFN-γ, but the secretion that presses down inflammatory Th2 factor IL-4 is not had influence (Fig. 7 A and B).This selectivity to the Th1 cell suppresses to change the ratio of IFN-γ/IL-4, has regulated the balance of Th1/Th2 subgroup in the body.From the T cellular immune mechanism of autoimmune disease morbidity, the CGRP gene therapy will suppress the generation of autoimmune inflammatory reaction.

6CGRP plasmid injection back is to the influence of type 1 diabetes sickness rate

We have compared the sickness rate of empty plasmid injection matched group and CGRP plasmid injection transgenic mice diabetes, and being higher than 12mmol/L with blood glucose is the onset diabetes standard.The CGRP transgenic has significantly reduced the sickness rate that STZ causes Autoimmune Diabetes as seen from Figure 8, reduces to 23% by 73% during 4 weeks.

7CGRP injection back is to the influence of diabetic mice plasma insulin level and blood sugar level

We measure and have compared the average blood sugar level of matched group and CGRP therapeutic transgene group mice again, see Fig. 9 A.Plasmid injection back CGRP treatment group blood glucose reduces than the empty plasmid matched group, continues more than 4 weeks, and the reduction amplitude reaches 3mmol/L during 4 weeks.

Adopt the measured by radioimmunoassay plasma insulin level.Fig. 9 B has compared the change of the plasma insulin of two groups of mices, and consistent with the result that blood glucose improves, CGRP treatment group insulin level significantly increases than the empty plasmid matched group.Persistent period also reached more than 4 weeks.The state of an illness that can significantly reduce the sickness rate of diabetes after the recombinant C GRP plasmid intramuscular injection that we make up and alleviate diabetes is described.

List of references:

1.Amara?SG，Jones?V，Rosenfeld?MG，Ong?ES，Evans?RM.AltemativeRNA?processing?in?calcitonin?gene?expression?generates?mRNAs?encodingdifferent?polypeptide?products.Nature，1982，298：240-244.

2.Xing?L，Guo?J，Wang?X.Induction?and?expression?of?β-calcitoningene-related?peptide?in?rat?T?lymphocytes?and?its?significance.J?Immunol，2000.165：4359-4366.

Sequence table

＜110〉Peking University

＜120〉a kind of plasmid that is used for direct intramuscular injection for treating diabetes

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Claims (1)

1. plasmid that is used for direct intramuscular injection for treating diabetes, this plasmid is the carrier pcDNA3.1 (-) that contains the CGRP gene, wherein, the CGRP gene has the nucleotide sequence shown in the SEQ ID NO:1, can express under the guiding of the promoter among the pcDNA3.1 (-).

Images