CN1238522C - Interleukin-2 and tes method of its gene in drug-stopping application - Google Patents
Interleukin-2 and tes method of its gene in drug-stopping application Download PDFInfo
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Abstract
The present invention discloses a detecting method of interleukin-2 (IL-2) and genes thereof in drug-stopping application, which belongs to the technical field of gene engineering. The method of the present invention indicates that IL-2 protein which is injected into the subarachnoid space has a favorable drug-stopping function, and the effects of the IL-2 protein are dependent on doses; pcDNA3-IL-2 genes also have a certain drug-stopping function, and the drug-stopping effects of the pcDNA3-IL-2 genes can be better and more suitable for practical use by further optimizing the lead-in conditions of the pcDNA3-IL-2 genes.
Description
Technical field:
The invention belongs to gene engineering technology field.Be specifically related to interleukin II (IL-2) and gene thereof the testing method in drug rehabilitation is used.
Background technology:
Drugs, not only serious harm human health, whole world drug addict has population more than 100,000,000, has brought a series of insoluble problems to society.To the able-bodied drugs of harm world people, national governments and scientists are just sparing no effort to study, develop to remove medicine and the method that drugs produce physiology/psychoreaction, make the drug addict reach the purpose of detoxification, de-addiction rapidly.But because special physiology and psychoreaction that drugs cause, fail so far to find a kind of drug addict's of making safety, effectively, no dependence ground breaks away from " miraculous cure " of drugs.Prior treatment method exists the side effect that is difficult to accept, and its application is also usually because habituation and limited.And whole work to the pharmacological dependence Journal of Sex Research still rests in the vicious circle of " new morphine compounds replaces old morphine compounds ", and " methadone successive subtraction method ", still used as " standard care " so far by developed country.Society presses for a kind of no habituation own, better control Withrawal symptom and the little medicine of toxic side effect is arranged.
The discovery of the analgesic activity of a kind of important lymphokine-interleukin II in the immunity system and the effect of control morphine abstinence syndrome symptom may provide an important instrument for us.China Liu Xin wall academician etc. finds that at first IL-2 has analgesic activity.Experiment shows that the existing central analgesia effect of IL-2 has the periphery analgesic activity again, and the clinical observation discovery has good analgesic effect after the not good terminal cancer pain patient of analgesic effect is used IL-2.Experiment finds that also the analgesic activity of IL-2 may be relevant with opiate receptor.Reach morphine on insensitive europathology pain model in the morphine tolerance, IL-2 has more significant analgesic activity than morphine.Because drug addict's immunologic hypofunction, IL-2 are expected to improve on the one hand body's immunological function, control Withrawal symptom on the one hand.Yet IL-2 active transformation period in body is very short, must heavy dose of continuous use, and the trouble of medical expenses height and long term injections, thus influenced the widespread use of result of treatment and this medicine.Gene therapy is to address this problem to have brought new hope, and it is a kind of method by transgenosis, will have the carrier of expressing goal gene and import in relevant cell and the tissue, makes the method for transcribing with translation product performance therapeutic action.The genophore that is used for gene therapy at present has virus vector and non-virus carrier.Liposome belongs to the latter, and the transgenosis safe of its mediation is good, is used in body (in vivo) gene therapy; The virus vector good stability, transgene efficiency height, but transfection division or somatoblast not; After adenovirus enters nucleus, form with episome (episome) exists, and does not integrate, and does not therefore have carcinogenic, the mutagenicity of retroviral vector potential, security is better, and it is neural in the body gene therapy that these characteristics have determined that adenovirus is very suitable for.After adenovirus carrier injects subarachnoid space, adenovirus enters pia mater spinalis and arachnoid membrane internal breeding, gene expression product acts on spinal cord and sensory nerve root with " paracrine pattern ", can regulate and control nerve in spinal levels, this gene therapy mode is worn and can be finished by waist, so be hopeful to be applied to out-patient's treatment.
Summary of the invention:
Technical problem to be solved by this invention is to design the testing method of IL-2 gene in drug rehabilitation is used, thereby can adopt IL-2 albumen, pcDNA3 plasmid-lipidosome or adenovirus vector-mediated IL-2 transgenosis to subarachnoid space, reach control morphine abstinence syndrome symptom, make the drug rehabilitation function of IL-2 reach the purpose of clinical application.
The invention provides a kind of interleukin II and gene thereof the testing method in drug rehabilitation is used, this method comprises the following steps:
(1) structure of eukaryon expression plasmid pcDNA3-IL-2
Human IL-2's gene is cut the human IL-2 cDNA (462bp) that the total length that obtains EcoR I and Xho I joint contains signal peptide through EcoR I and Xho I enzyme and is cloned into the pcDNA3 carrier of cutting through same enzyme (Invitrogen).The IL-2 gene is expressed under the control of CMV promotor, and the plasmid of structure is transformed into the e. coli jm109 amplification, and alkaline lysis prepares plasmid, the polyethylene glycol precipitation purifying.Determine plasmid concentration by the photoabsorption of measuring 260nm, plasmid is diluted in the phosphoric acid buffer (PB) that contains 5% glucose during injection;
(2) structure of recombinant adenovirus Ad5-IL-2
Human IL-2's gene is cut the human IL-2 cDNA (462bp) that the total length that obtains EcoR I and Xho I joint contains signal peptide through EcoR I and Xho I enzyme and is cloned into the pCA13 carrier of cutting through same enzyme (Microbix).Obtain pCA13-IL-2, this moment, the IL-2 gene was expressed under the control of CMV promotor, pCA13-IL-2 and adenoviral gene group plasmid pBHG10 (Microbix) cotransfection 293 cells, carry out homologous recombination, the screening recombinant adenovirus, the amplification purification adenovirus, viral dilution is in the PBS that contains 5% sucrose during injection;
(3) morphine relies on the foundation of (habituation) mouse model
Form morphine-dependent mice model (this group mouse is a morphine dependence group) with incremental dose method.Morphine-dependent mice abdominal injection naloxone 4mg.kg
-1Can excite Withrawal symptom, this group mouse is the morphine abstinence syndrome group;
(4) morphine relies on the foundation of rat model
Form morphine with incremental dose method and rely on rat model (this group rat is a morphine dependence group).Morphine relies on rats by intraperitoneal injection naloxone 4mg.kg
-1Can excite Withrawal symptom, this group rat is the morphine abstinence syndrome group;
(5) foundation of the plasmid-mediated IL-2 gene inhibition of IL-2 albumen and pcDNA3 mouse morphine abstinence syndrome symptom testing method
With the morphine addiction mouse model that incremental dose method is set up, after 3 hours the mouse of habituation is divided into 8 groups at random, 10 every group for morphine at last.The protein for treatment group: (subarachnoid space) injects 5 * 10 respectively between the 5th and the 6th lumbar vertebrae
3, 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4IU IL-2 albumen, control group are the 0.01M phosphoric acid buffer (PB) that contains 5% glucose.IL-2 gene therapy group: 8 μ g pcDNA3/8 μ g lipofectamine (control group, injection in 24 hours in advance), 8 μ g pcDNA3-IL-2/8 μ g lipofectamine (gene therapy group, injection in 24 hours in advance).The injection cumulative volume is 10 μ l.Injected back 5 minutes, each organizes mouse peritoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe mouse occurs in the latent period, 15 minutes that mouse begins to jump number of skips and the body weight change of mouse in 30 minutes;
(6) IL-2 albumen suppresses the foundation of rat morphine abstinence syndrome symptom testing method
With the morphine-addicted rats model that incremental dose method is set up, give morphine after 3 hours at last, the rat of habituation is divided into 4 groups at random, every group of 5-11 only, (subarachnoid space) injects 2 * 10 respectively in the insertion catheter sheath between the 5th and the 6th lumbar vertebrae
4, 4 * 10
4, 1.4 * 10
5IU IL-2 albumen, control group are the PB that contains 5% glucose, and the injection cumulative volume is 30 μ l.Injected back 5 minutes, each organizes rats by intraperitoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe the various Withrawal symptoms of rat, as the wet shake of dog sample, abnormal posture, excitation, grit one's teeth, diarrhoea, hydrostomia, lose weight, and calculate Withrawal symptom general comment score value;
(7) foundation of adenovirus vector-mediated IL-2 gene inhibition mouse morphine abstinence syndrome symptom testing method
Rat inserts in the catheter sheath (subarachnoid space) and injects the PBS that contains 5% sucrose, 1 * 10 respectively between the 5th and the 6th lumbar vertebrae
7Pfu adenovirus 5 types (Ad5), 1 * 10
7PfuAd5-IL-2.The injection cumulative volume is 10 μ l.One week of injection back is measured abdominal injection naloxone 4mg.kg
-1The Withrawal symptom that excites is observed mouse occurs in the latent period, 15 minutes that mouse begins to jump number of skips and the body weight change of mouse in 30 minutes;
(8) habituation test
A. the perpendicular tail test of mouse: mouse is divided into six groups at random, and 10 every group, difference abdominal injection morphine 20mg.kg
-1(positive control), abdominal injection contain the PB (negative control) of 5% glucose, abdominal injection IL-2 albumen 1 * 10
5IU or 1 * 10
6IU, and subarachnoid injection IL-2 albumen 1 * 10
4IU or 1 * 10
5IU.Observing after the administration in 2 hours mouse has or not and S shape straub tail reaction occurs;
B. mouse jump reaction test: mouse is divided into three groups at random, every group 10, subcutaneous injection contains the PB (negative control group) or the morphine (positive controls) of 5% glucose respectively, abdominal injection IL-2 albumen, 5 times (9:00,10:00,11:00,13:00,15:00) of injection in first day, 2 times (9:00,11:00) of injection in second day.Morphine group mouse initial dose is 2.5mg.kg
-1, after this by 5,10,20,30,40,50mg.kg
-1Dosage escalation; IL-2 protein groups dosage is 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4, 1.6 * 10
5, 3.2 * 10
5And6.4 * 10
5IU.After last administration 2 hours, abdominal injection naloxone 10mg.kg
-1, observe mouse jump reaction times in 10 minutes;
(9) statistical method
Adopt one-way analysis of variance.
Method result of study of the present invention:
(1) subarachnoid injection IL-2 albumen suppresses mouse morphine abstinence syndrome symptom
Subarachnoid injection IL-2 albumen (1 * 10
4IU) can obviously prolong the latent period (p＜0.01) that mouse begins to jump, and obviously suppress the number of skips (p＜0.01) that mouse occurs, its effect is dose-dependently; IL-2 albumen (2 * 10
4IU) can obviously suppress lose weight (p＜0.05) (Fig. 1, A, B, C) of mouse.
(2) subarachnoid injection IL-2 gene (pcDNA3-IL-2) suppresses mouse morphine abstinence syndrome symptom
8 μ g pcDNA3-IL-2/8 μ g lipofectamine can obviously suppress the body weight change (p＜0.05) of mouse, and can prolong the latent period (p＜0.01) that mouse begins to jump and suppress the number of skips (p＜0.01) (Fig. 1, A, B, C) that mouse occurs.
(3) subarachnoid injection IL-2 albumen suppresses rat morphine abstinence syndrome symptom
Subarachnoid injection IL-2 albumen can obviously suppress the total score value of Withrawal symptom (p＜0.05) of rat, comprises abnormal posture, excitation, diarrhoea, hydrostomia, loses weight; Wet dog sample shake, the score value of gritting one's teeth there are the tendency of minimizing, but no statistical significance.The long and shows that IL-2 albumen also can obviously suppress the most morphine abstinence syndrome symptom of rat.
(4) IL-2 albumen does not have habituation
A. the perpendicular tail test of mouse: S shape straub tail reaction all appears in morphine group mouse, and excitement is run, and IL-2 protein groups mouse does not have straub tail reaction, and is movable normal.
B. mouse jump reaction test: IL-2 protein groups (0.9 ± 0.5) is more close with control group (0.4 ± 0.3), with morphine group (14.1 ± 1.6) difference (p＜0.01) of highly significant is arranged more then.
Table 1. subarachnoid injection IL-2 albumen is to the influence of rat morphine abstinence syndrome symptom
After forming morphine dependence rat model, (subarachnoid space) injects 2 * 10 respectively in the insertion catheter sheath between the 5th and the 6th lumbar vertebrae
4, 4 * 10
4, 1.4 * 10
5IU IL-2 albumen, control group (vehicle) is for containing the PB of 5% glucose, and the injection cumulative volume is 30 μ l.Injected back 5 minutes, each organizes rats by intraperitoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe the various Withrawal symptoms of rat, as the wet shake of dog sample, abnormal posture, excitation, grit one's teeth, diarrhoea, hydrostomia, lose weight, and calculate Withrawal symptom general comment score value.Statistical significance is decided to be
*P＜0.05 (comparing) with vehicle.
Table 1
| IL-2 dosage | 2.0×10 4IU | 4.0×10 4IU | 1.4×10 5IU | vehicle |
| The wet dog sample shake of the number of animals motility disease score value abnormal posture salivation non-motility disease score value diarrhoea excitation general comment score value of losing weight of gritting one's teeth | 5 1.60±0.75 6.40±1.60 1.20±0.49 0.20±0.20 5.60±0.98 1.60±1.60 10.00±1.58 26.60±3.01 * | 5 0.80±0.49 6.80±0.49 0±0 * 0.10±0.10 3.20±1.50 * 0.8±0.49 * 6.00±1.00 * 17.70±1.74 * | 5 0±0 5.6±0.75 * 0.4±0.4 * 0.0±0 2.4±1.6 * 0.4±0.4 * 7.00±1.22 * 15.8±3.43 * | 11 1.27±0.49 8.00±0 1.82±0.12 0.27±0.10 7.27±0.49 3.54±0.68 11.82±1.02 33.95±1.06 |
After 7 IL-2 albumen of table 2. injected in mice (abdominal cavity) or the morphine (subcutaneous), abdominal injection naloxone 4mg.kg
-1The mouse jump that excites
Mouse subcutaneous injection respectively contains the PB (negative control group) or the morphine (positive controls) of 5% glucose, abdominal injection IL-2 albumen, 5 times (9:00,10:00,11:00,13:00,15:00) of injection in first day, 2 times (9:00,11:00) of injection in second day.Morphine group mouse initial dose is 2.5mg.kg
-1, after this by 5,10,20,30,40,50mg.kg
-1Dosage escalation; IL-2 protein groups dosage is 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4, 1.6 * 10
5, 3.2 * 10
5And 6.4 * 10
5IU.After last administration 2 hours, abdominal injection naloxone 10mg.kg
-1, observe mouse jump reaction times in 10 minutes.Statistical significance is decided to be
*P＜0.05 (comparing) with the morphine group.
Table 2
| Medicine and route of administration | Number of skips (mouse/sum of jump) |
| PB (subcutaneous injection) morphine (subcutaneous injection) the IL-2 albumen (abdominal injection) that contains 5% glucose | 0.4±0.3 *(2/10) 14.1±1.6(10/10) 0.9±0.5 *(3/10) |
Description of drawings:
The influence of Fig. 1 (A, B, C) subarachnoid injection IL-2 albumen and pcDNA3-IL-2 gene pairs mouse morphine abstinence syndrome symptom
After forming the morphine-dependent mice model, (subarachnoid space) injected respectively between the 5th and the 6th lumbar vertebrae: 5 * 10
3, 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4IU IL-2 albumen, control group (vehicle) is for containing the PB of 5% glucose, n=9-18; The gene therapy group is 8 μ g pcDNA3-IL-2/8 μ g lipofectamine (injections in 24 hours in advance), and its control group is 8 μ g pcDNA3/8 μ g lipofectamine (injections in 24 hours in advance), n=10.The injection cumulative volume is 10 μ l.Injected back 5 minutes, each organizes mouse peritoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, statistics is respectively organized latent period (second) that mouse begins to jump (Figure 1A) and the number of skips (Figure 1B) of mouse appearance in 15 minutes and mouse body weight change (gram) (Fig. 1 C) in 30 minutes respectively, X-coordinate is the IL-2 concentration of subarachnoid injection among the figure, ordinate zou be respectively latent period (second) (Figure 1A), number of skips (Figure 1B) and body weight change (gram) (Fig. 1 C), statistical significance is decided to be
*P＜0.05,
*P＜0.01 (comparing) with vehicle;
#P＜0.05,
##P＜0.01 (comparing) with 8 μ g pcDNA3/8 μ g lipofectamine.
Annotate: 1. control group (vehicle)
2.5 * 10
3IU IL-2 albumen
3.1 * 10
4IU IL-2 albumen
4.2 * 10
4IU IL-2 albumen
5.4 * 10
4IU IL-2 albumen
6.8 * 10
4IU IL-2 albumen
7.8μg pcDNA3-IL-2/8μg lipofectamine
8.8μg pcDNA3/8μg lipofectamine
Show by test method results of the present invention: subarachnoid injection IL-2 albumen has drug treatment function preferably, and its effect is dose-dependently.Be characterized in that itself does not have habituation, and enhancing body's immunological function simultaneously, this drug addict to hypoimmunity is very useful, and habituation not again can raise immunity, be present any methods of treatment can't accomplish.The pcDNA3-IL-2 gene also has certain drug treatment function, and its effect is poorer than high dosage IL-2 albumen, may be because the proteic instantaneous secretory volume of IL-2 is lower.But then, because pcDNA3-IL-2 expressed IL-2 albumen sustainable 6 days (the proteic transformation period of IL-2 than injection prolongs about 300 times), be very convenient, economical to whole withdrawal.By the pcDNA3-IL-2 gene being imported the further optimization of condition, its drug abstinence is expected better to be more suitable for practical application.
Embodiment:
(1) structure of eukaryon expression plasmid pcDNA3-IL-2
Human IL-2's gene is cut the human IL-2 cDNA (462bp) that the total length that obtains EcoR I and Xho I joint contains signal peptide through EcoR I and Xho I enzyme and is cloned into the pcDNA3 carrier of cutting through same enzyme (Invitrogen).The IL-2 gene is expressed under the control of CMV promotor, and the plasmid of structure is transformed into the e. coli jm109 amplification, and alkaline lysis prepares plasmid, the polyethylene glycol precipitation purifying.Determine plasmid concentration by the photoabsorption of measuring 260nm.Plasmid is diluted in the phosphoric acid buffer (PB) that contains 5% glucose during injection.
(2) structure of recombinant adenovirus Ad5-IL-2
Human IL-2's gene is cut the human IL-2 cDNA (462bp) that the total length that obtains EcoR I and Xho I joint contains signal peptide through EcoR I and Xho I enzyme and is cloned into the pCA13 carrier of cutting through same enzyme (Microbix).Obtain pCA13-IL-2, this moment, the IL-2 gene was expressed under the control of CMV promotor, and pCA13-IL-2 and adenoviral gene group plasmid pBHG10 (Microbix) cotransfection 293 cells carry out homologous recombination, the screening recombinant adenovirus.The amplification purification adenovirus, viral dilution is in the PBS that contains 5% sucrose during injection.
(3) foundation of morphine-dependent mice model
Form morphine-dependent mice model (this group mouse is a morphine dependence group) with incremental dose method.Morphine-dependent mice abdominal injection naloxone 4mg.kg
-1Can excite Withrawal symptom, this group mouse is the morphine abstinence syndrome group.
(4) morphine relies on the foundation of rat model
Form morphine with incremental dose method and rely on rat model (this group rat is a morphine dependence group).Morphine relies on rats by intraperitoneal injection naloxone 4mg.kg
-1Can excite Withrawal symptom, this group rat is the morphine abstinence syndrome group.
(5) foundation of the plasmid-mediated IL-2 gene inhibition of IL-2 albumen and pcDNA3 mouse morphine abstinence syndrome symptom testing method
With the morphine addiction mouse model that incremental dose method is set up, give morphine after 3 hours at last, the mouse of habituation is divided into 8 groups, 10 every group at random.The protein for treatment group: (subarachnoid space) injects 5 * 10 respectively between the 5th and the 6th lumbar vertebrae
3, 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4IU IL-2 albumen, control group are the 0.01M phosphoric acid buffer (PB) that contains 5% glucose.IL-2 gene therapy group: 8 μ g pcDNA3/8 μ g lipofectamine (control group, injection in 24 hours in advance), 8 μ gpcDNA3-IL-2/8 μ g lipofectamine (gene therapy group, injection in 24 hours in advance).The injection cumulative volume is 10 μ l.Injected back 5 minutes, each organizes mouse peritoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe mouse occurs in the latent period, 15 minutes that mouse begins to jump number of skips and the body weight change of mouse in 30 minutes.
(6) IL-2 albumen suppresses the foundation of rat morphine abstinence syndrome symptom testing method
With the morphine-addicted rats model that incremental dose method is set up, give morphine after 3 hours at last, the rat of habituation is divided into 4 groups at random, every group of 5-11 only, (subarachnoid space) injects 2 * 10 respectively in the insertion catheter sheath between the 5th and the 6th lumbar vertebrae
4, 4 * 10
4, 1.4 * 10
5IU IL-2 albumen, control group are the PB that contains 5% glucose, and the injection cumulative volume is 30 μ l.Injected back 5 minutes, each organizes rats by intraperitoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe the various Withrawal symptoms of rat, as the wet shake of dog sample, abnormal posture, excitation, grit one's teeth, diarrhoea, hydrostomia, lose weight, and calculate Withrawal symptom general comment score value.
(7) foundation of adenovirus vector-mediated IL-2 gene inhibition mouse morphine abstinence syndrome symptom testing method
Rat inserts in the catheter sheath (subarachnoid space) and injects the PBS that contains 5% sucrose, 1 * 10 respectively between the 5th and the 6th lumbar vertebrae
7Pfu adenovirus 5 types (Ad5), 1 * 10
7PfuAd5-IL-2.The injection cumulative volume is 10 μ l.One week of injection back is measured abdominal injection naloxone 4mg.kg
-1The Withrawal symptom that excites is observed mouse occurs in the latent period, 15 minutes that mouse begins to jump number of skips and the body weight change of mouse in 30 minutes.
(8) habituation test
A. the perpendicular tail test of mouse: mouse is divided into six groups at random, and 10 every group, difference abdominal injection morphine 20mg.kg
-1(positive control), abdominal injection contain the PB (negative control) of 5% glucose, abdominal injection IL-2 albumen 1 * 10
5IU or 1 * 10
6IU, and subarachnoid injection IL-2 albumen 1 * 10
4IU or 1 * 10
5IU.Observing after the administration in 2 hours mouse has or not and S shape straub tail reaction occurs.
B. mouse jump reaction test: mouse is divided into three groups at random, every group 10, subcutaneous injection contains the PB (negative control group) or the morphine (positive controls) of 5% glucose respectively, abdominal injection IL-2 albumen, 5 times (9:00,10:00,11:00,13:00,15:00) of injection in first day, 2 times (9:00,11:00) of injection in second day.Morphine group mouse initial dose is 2.5mg.kg
-1, after this by 5,10,20,30,40,50mg.kg
-1Dosage escalation; IL-2 protein groups dosage is 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4, 1.6 * 10
5, 3.2 * 10
5And6.4 * 10
5IU.After last administration 2 hours, abdominal injection naloxone 10mg.kg
-1, observe mouse jump reaction times in 10 minutes.
(9) statistical method
Adopt one-way analysis of variance.
Claims (1)
1, interleukin II IL-2 and gene thereof the testing method in drug rehabilitation is used is characterized in that this method comprises the following steps:
(1) structure of eukaryon expression plasmid pcDNA3-IL-2
Human IL-2's gene is cut the human IL-2 cDNA 462bp that the total length that obtains EcoR I and Xho I joint contains signal peptide through EcoR I and Xho I enzyme and is cloned into the pcDNA3 carrier Invitrogen that cuts through same enzyme, the IL-2 gene is expressed under the control of CMV promotor, the plasmid that makes up is transformed into the e. coli jm109 amplification, alkaline lysis prepares plasmid, the polyethylene glycol precipitation purifying, determine plasmid concentration by the photoabsorption of measuring 260nm, plasmid is diluted in the phosphoric acid buffer that contains 5% glucose during injection;
(2) structure of recombinant adenovirus Ad5-IL-2
Human IL-2's gene is cut the human IL-2 cDNA 462bp that the total length that obtains EcoR I and Xho I joint contains signal peptide through EcoR I and Xho I enzyme and is cloned into the pCA13 carrier Microbix that cuts through same enzyme, obtain pCA13-IL-2, this moment, the IL-2 gene was expressed under the control of CMV promotor, pCA13-IL-2 and adenoviral gene group plasmid pBHG10 Microbix cotransfection 293 cells, carry out homologous recombination, the screening recombinant adenovirus, the amplification purification adenovirus, viral dilution is in the PBS that contains 5% sucrose during injection;
(3) foundation of morphine-dependent mice model
Form the morphine-dependent mice model with incremental dose method, this group mouse is a morphine dependence group, morphine-dependent mice abdominal injection naloxone 4mg.kg
-1Can excite Withrawal symptom, this group mouse is the morphine abstinence syndrome group;
(4) morphine relies on the foundation of rat model
Form morphine with incremental dose method and rely on rat model, this group rat is a morphine dependence group, and morphine relies on rats by intraperitoneal injection naloxone 4mg.kg
-1Can excite Withrawal symptom, this group rat is the morphine abstinence syndrome group;
(5) foundation of the plasmid-mediated IL-2 gene inhibition of IL-2 albumen and pcDNA3 mouse morphine abstinence syndrome symptom testing method
With the morphine addiction mouse model that incremental dose method is set up, give morphine after 3 hours at last, the mouse of habituation is divided into 8 groups at random, 10 every group, the protein for treatment group: between the 5th and the 6th lumbar vertebrae, subarachnoid space injects 5 * 10 respectively
3, 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4IU IL-2 albumen, control group is the 0.01M phosphoric acid buffer that contains 5% glucose, IL-2 gene therapy group: 8 μ g pcDNA3/8 μ g lipofectamine, control group, injection in 24 hours in advance, 8 μ g pcDNA3-IL-2/8 μ g lipofectamine, the gene therapy group, injection in 24 hours in advance, the injection cumulative volume is 10 μ l, injected back 5 minutes, each organizes mouse peritoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe mouse occurs in the latent period, 15 minutes that mouse begins to jump number of skips and the body weight change of mouse in 30 minutes;
(6) IL-2 albumen suppresses the foundation of rat morphine abstinence syndrome symptom testing method
With the morphine-addicted rats model that incremental dose method is set up, give morphine after 3 hours at last, the rat of habituation is divided into 4 groups at random, every group of 5-11 only, in the insertion catheter sheath, subarachnoid space injects 2 * 10 respectively between the 5th and the 6th lumbar vertebrae
4, 4 * 10
4, 1.4 * 10
5IU IL-2 albumen, control group are the PB that contains 5% glucose, and the injection cumulative volume is 30 μ l, injects back 5 minutes, and each organizes rats by intraperitoneal injection naloxone 4mg.kg
-1Excite Withrawal symptom, observe the various Withrawal symptoms of rat, as the wet shake of dog sample, abnormal posture, excitation, grit one's teeth, diarrhoea, hydrostomia, lose weight, and calculate Withrawal symptom general comment score value;
(7) foundation of adenovirus vector-mediated IL-2 gene inhibition mouse morphine abstinence syndrome symptom testing method
Rat inserts between the 5th and the 6th lumbar vertebrae in the catheter sheath, subarachnoid space, and injection contains the PBS of 5% sucrose, 1 * 10 respectively
7Pfu adenovirus 5 type Ad5,1 * 10
7Pfu Ad5-IL-2, the injection cumulative volume is 10 μ l, one week of injection back is measured abdominal injection naloxone 4mg.kg
-1The Withrawal symptom that excites is observed mouse occurs in the latent period, 15 minutes that mouse begins to jump number of skips and the body weight change of mouse in 30 minutes;
(8) habituation test
A. the perpendicular tail test of mouse: mouse is divided into six groups at random, and 10 every group, difference abdominal injection morphine 20mg.kg
-1Positive control, abdominal injection contain the PB negative control of 5% glucose, abdominal injection IL-2 albumen 1 * 10
5IU or 1 * 10
6IU, and subarachnoid injection IL-2 albumen 1 * 10
4IU or 1 * 10
5IU observes after the administration in 2 hours mouse and has or not and S shape straub tail reaction occurs;
B. mouse jump reaction test: mouse is divided into three groups at random, every group 10, subcutaneous injection contains the PB negative control group or the morphine positive controls of 5% glucose respectively, abdominal injection IL-2 albumen, injection in first day 5 times, 9:00,10:00,11:00,13:00,15:00, injection in second day 2 9:00,11:00, morphine group mouse initial dose is 2.5mg.kg
-1, after this by 5,10,20,30,40,50mg.kg
-1Dosage escalation; IL-2 protein groups dosage is 1 * 10
4, 2 * 10
4, 4 * 10
4, 8 * 10
4, 1.6 * 10
5, 3.2 * 10
5And 6.4 * 10
5IU, after last administration 2 hours, abdominal injection naloxone 10mg.kg
-1, observe mouse jump reaction times in 10 minutes;
(9) statistical method
Adopt one-way analysis of variance.
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