CN104189918A - Application of dynein light chain roadblock-type 2 in preparation of medicine treating kidney failure and the medicine thereof - Google Patents
Application of dynein light chain roadblock-type 2 in preparation of medicine treating kidney failure and the medicine thereof Download PDFInfo
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- CN104189918A CN104189918A CN201410348885.4A CN201410348885A CN104189918A CN 104189918 A CN104189918 A CN 104189918A CN 201410348885 A CN201410348885 A CN 201410348885A CN 104189918 A CN104189918 A CN 104189918A
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Abstract
The invention discloses an application of a compound inhibiting dynein light chain roadblock-type 2 in preparation of a medicine treating kidney diseases especially kidney failure diabetes. A knockdown dynein light chain roadblock-type 2 plasmid is used for preparing the medicine. A medicine for treating diabetic nephropathy is also disclosed and comprises the knockdown dynein light chain roadblock-type 2 plasmid. The medicine can be used for achieving novel, safe, effective and controllable gene therapy methods and for inhibiting TGF-beta mediated kidney fibrosis.
Description
Technical field
The present invention relates to application and the medicine thereof of dynein light chain roadblock-2 type preparation treatment renal failure medicine.
Background technology
Renal fibrosis causes renal failure in latter stage at end, due to the expression of the extracellular matrix of TGF-beta/Smad signals-modulating in kidney disease, so be to control a Fibrotic attractive Therapeutic Method for TGF-beta signal.Yet, in the complexity of TGF-beta signal transduction path, make to select suitable treatment target spot difficulty.Especially, for the method for the individual element in TGF-beta signal transduction path, be proved to be effectiveness little.
In the past ten years, many strategies are developed directly to block TGF-beta impact, comprise and use the neutralizing antibody of TGF-beta, the oligodeoxynucleotide of constrain TGF-beta (ODN), solubility mankind TGF-beta II receptor, or the specific inhibitor of TGF-beta receptor kinase.Yet, after investigating further, find to employ these tactics and make not produce as expected effectively result.This may be can damage the effective anti-inflammatory characteristic of TGF-beta owing to blocking TGF-beta signal upstream pathway, and promotes subsequently kidney inflammation.On the other hand, crosstalking of intracellular Smad path can be hindered the strategy of targeting TGF-beta and TGF-beta receptor kinase.
Summary of the invention
Main purpose of the present invention is the deficiency for the medicine of existing treatment nephropathy, provides dynein light chain roadblock-2 type to treat nephropathy as the application in renal failure diabetes medicament and medicine thereof in preparation.
For achieving the above object, the present invention is by the following technical solutions:
By suppressing the application of the material of dynein light chain roadblock-2 type for the preparation for the treatment of nephropathy diabetes medicament, wherein use to strike low dynein light chain roadblock-2 type plasmid, make described medicine.
Preferably:
Described in inciting somebody to action, strike low dynein light chain roadblock-2 type plasmid and mix to make described medicine with microbubble contrast agent.
Described microbubble contrast agent is sulfur hexafluoride microbubble contrast agent, preferably
contrast agent (Bracco imaging B.V, Plan-Les-Quates Geneve, Switzerland).
Described volume ratio of striking low dynein light chain roadblock-2 type plasmid and described microbubble contrast agent is 1:1.
The plasmid that structure strikes low dynein light chain roadblock-2 type is that a short hairpin RNA (shRNA) for dynein light chain roadblock-2 type messenger RNAs (messenger RNA) is synthesized in carrier, to produce the bobby pin RNA of dynein light chain roadblock-2 type, wherein, the sequence of described short hairpin RNA is (GGA CAA CTC AAC AAC TGT TCA ATA T).Preferably, described carrier is pSilencer4.1 carrier (Invitrogen, Carlsbad, VA, USA).
Do not adopt and strike low dynein light chain roadblock-2 type plasmid described in endotoxic plasmid kit generation.
Make the intravenous injection of the microvesicle of the shRNA plasmid that strikes low dynein light chain roadblock-2 type that contains 1 μ M/ml.
Treat a medicine for nephropathy diabetes, contain and strike low dynein light chain roadblock-2 type plasmid.
Also contain microbubble contrast agent, preferably, described in strike low dynein light chain roadblock-2 type plasmid and described microbubble contrast agent volume ratio be 1:1.
Adopt the intravenous injection containing the shRNA plasmid that strikes low dynein light chain roadblock-2 type of 1 μ M/ml.
Beneficial effect of the present invention:
The invention provides and suppress dynein light chain-2 type to treat medicine of kidney disease and preparation method thereof.Dynein light chain roadblock-2 type affects the activation of Smad3 in kidney disease process.Therefore the activation that the phosphorylation mechanism that, suppresses the expression specificity compacting Smad3 albumen of dynein light chain-2 type by this removes Smad3.In addition, we have proved and have suppressed expression and α 2 (I) the collagen promoter activity that dynein light chain-2 type has also reduced NTx.We also find, the expression of expression inhibiting α-SMA in renal cells fibrotic processes of compacting dynein light chain-2 type, this may be to abolish the activation of autocrine TGF-beta signal path in renal fibrosis process because suppress dynein light chain-2 type.
Result shows, suppresses dynein light chain-2 type and suppresses TGF-beta signal downstream pathway and stop excessive extracellular matrix to generate, and the inhibition of dynein light chain-2 type will be a kind of effective treatment of hitting chronic kidney disease.
Owing to adopting the gene therapy of this medicine can directly suppress the activation of TGF-beta/Smad3 signal conduction in kidney lysis, and renal fibrosis in minimizing body, aspect short application use, adopt the gene therapy of this medicine can suppress to be directly implemented in chronic kidney disease patient, go to end the progress of renal fibrosis, improve the performance of renal function.On long terms, this gene therapy can be used in the generation that prevents kidney disease, by suppressing TGF-beta signal at any abnormal activation of kidney, reduces the appearance of renal fibrosis.
Medicine provided by the invention can be used for realizing the renal fibrosis that a kind of novel, safe, effective, controllable gene therapy suppresses TGF-beta mediation.
Accompanying drawing explanation
PCR in real time testing result when Figure 1A illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention;
Immunoblotting result when Figure 1B illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention;
Immunohistochemistry result when Fig. 1 C illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention;
Immunochemistry result after quantification when Fig. 1 D illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention;
Histology and immunohistochemical result when Fig. 2 A illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention;
PCR in real time testing result when Fig. 2 B illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention;
Western blotting result when Fig. 2 C illustrates the medicine that does not use the embodiment of the present invention and after the medicine of the use embodiment of the present invention.
The specific embodiment
Below in conjunction with accompanying drawing, embodiments of the present invention are elaborated.Should be emphasized that, following explanation is only exemplary, rather than in order to limit the scope of the invention and to apply.
Smad3 is found in research, HSP72, and HSP90 specific inhibitor can reduce renal fibrosis.Therefore can provide the new way of the replacement therapy of renal fibrosis.Use may provide a kind of new, effective, safe treatment for nephropathy for the inhibitor of TGF-beta/Smad3 signal pathway.
Embodiments of the invention preferably adopt ultrasonic microbubble plasmid transfection technology by the shRNA plasmid transfection that suppresses dynein light chain-2 type to kidney, downward dynein light chain-2 type is in the expression of kidney cell.Can detect the good result that suppresses dynein light chain-2 type treatment diabetic nephropathy.
Specifically, in embodiment, be preferably provided for the medicine of the intravenous microvesicle that contains the shRNA plasmid (1 μ M/ml) that suppresses dynein light chain-2 type, preferred content 1 μ M/ml.Can, by this medicine plasmid of intravenous injection, then on kidney side, add ultrasonic energy (1W, 1MHz) 5 minutes.Ultrasonic therapeutic strengthens plasmid and is delivered to whole kidney, and efficiency can reach the nucleus of 90% cell glomerule, renal tubular interstitium and blood vessel wall.
the constituent of medicine
In a preferred embodiment, medicine comprises:
1. strike (KD plasmid) plasmid of low dynein light chain roadblock-2 types (DYNLRB2);
2.
(sulfur hexafluoride microvesicle) contrast agent (Bracco imaging B.V, Plan-Les-Quates Geneve, Switzerland).
In a preferred embodiment, also further provide the intravenous drug that contains said medicine composition.
the preparation of medicine
In a preferred embodiment, medicine adopt strike low dynein light chain roadblock-2 type plasmid and
(sulfur hexafluoride microvesicle) contrast agent mixes to prepare.
In preferred embodiment, the plasmid that structure strikes low dynein light chain roadblock-2 type is that a short hairpin RNA (shRNA) for dynein light chain roadblock-2 type messenger RNAs (messenger RNA) is synthesized to (Invitrogen in pSilencer4.1 carrier, Carlsbad, VA, USA), to produce the bobby pin RNA of dynein light chain roadblock-2 type, wherein, the sequence of described short hairpin RNA is (GGA CAA CTC AAC AAC TGT TCA ATA T).
And can not adopt containing endotoxic plasmid kit (Qiagen, Valencia, CA, USA) and remove a large amount of plasmids that strike low dynein light chain roadblock-2 type that produce.Finally, this plasmid preferably with the ratio of 1:1 (volume/volume) with
(sulfur hexafluoride microvesicle) contrast agent (Bracco imaging B.V, Plan-Les-Quates Geneve, Switzerland) mixing for standby use.
insecticide-applying way
1. intravenous injection contains the microvesicle (400 μ l) that structure strikes the plasmid of low dynein light chain roadblock-2 type.
2. on kidney side, add ultrasonic energy (1MHz, 1W) 5 minutes.
illustrating of curative effect of medication experiment and experimental data
For the expression of the verifying constrain dynein light chain roadblock-2 type effect to the beta induced renal fibrosis of TGF-in vivo, we utilize the one-sided urethral obstruction of mice (unilateral ureter obstruction, UUO) model is as the model of chronic nephropathy, adopt ultrasonic microbubble plasmid transfection technology by suppress dynein light chain roadblock-2 type plasmid transfection to kidney, lower dynein light chain roadblock-2 type in the expression of kidney cell, detect the effect of dynein light chain roadblock-2 type treatment chronic kidney disease.The preferred deal of plasmid in drug administration: 150 μ g/25g (plasmid/Mouse Weight).
We first set up the mice chronic kidney disease model of one-sided urethral obstruction, on the same day, our intravenous injection contains and strikes plasmid (the 1 μ M/ml of low dynein light chain roadblock-2 type, 400 μ l) microvesicle is to mice, then at the other ultrasonic energy (1W that adds of kidney, 1MHz's) 5 minutes, ultrasonic therapeutic strengthens plasmid and is delivered to whole kidney.
After one-sided urethral obstruction the 7th day, the expression of dynein light chain roadblock-2 type in kidney significantly increases (Figure 1A).Yet the plasmid that low dynein light chain roadblock-2 type is struck in input has suppressed the expression (Figure 1A) in kidney internally-powered albumen light chain roadblock-2 type of the mouse model of one-sided urethral obstruction nephropathy.
We study and strike the roadblock-2 type treatment of low dynein light chain and can whether affect TGF-signal beta, because TGF-beta signal is the key signal path of renal fibrosis, in the mouse model of one-sided urethral obstruction nephropathy, TGF-beta signal can be activated, increase the Connective Tissue Growth Factor (CTGF) of kidney and the expression of phosphorylation Smad3 (P-Smad3) albumen (Figure 1B, C).Yet plasmid inhibition TGF-β 1, the CTGF of low dynein light chain roadblock-2 type and the expression (Figure 1B, C) of phosphorylation Smad3 albumen are struck in input.This shows, strikes the activation of the TGF-signal beta in the expression inhibiting kidney of low dynein light chain roadblock-2 type, causes the inhibition of renal fibrosis.
Histology is upper, after one-sided urethral obstruction, within the 7th day, causes interstitial fibrosis and renal tubules atrophy (Fig. 2 A).When relatively, with the mice after empty carrier treatment, strike the type treatment of low dynein light chain roadblock-2 and reduce renal tubules atrophy and interstitial fibrosis (Fig. 2 A).Meet injury of kidney and interstitial fibrosis degree, ((Fig. 2 A-C) all raised in the expression that comprises type i collagen and α-smooth muscle actin (α-SMA), but the expression (Fig. 2 A-C) that the plasmid of low dynein light chain roadblock-2 type is lowered Fibrosis Markers is struck in input in the expression of Fibrosis Markers.These results have proved the interstitial fibrosis of the expression minimizing chronic kidney disease of striking low dynein light chain roadblock-2 type.
As shown in Figure 1A, PCR in real time testing result shows, after one-sided urethral obstruction the 7th day, and dynein light chain roadblock-2 type in kidney is expressed and is increased, but after input strikes the plasmid of low dynein light chain roadblock-2 type and suppress, it is just expressed and reduces.
As shown in Figure 1B, immunoblotting result shows, after one-sided urethral obstruction the 7th day, the expression of the Connective Tissue Growth Factor of kidney (CTGF) and phosphorylation Smad3 (P-Smad3) albumen increases, but with striking this increase of minimizing after the type plasmid treatment of low dynein light chain roadblock-2.
Immunochemistry result after quantification shown in immunohistochemistry result shown in Fig. 1 C and Fig. 1 D shows, after one-sided urethral obstruction the 7th day, the expression of the phosphorylation Smad3 of kidney (P-Smad3) albumen increases, but reduces this increase after striking the type plasmid treatment of low dynein light chain roadblock-2.
As shown in Figure 2 A, from histology and immunohistochemical result, all show, at one-sided urethral obstruction after 7 days, strike the type plasmid treatment of low dynein light chain roadblock-2 and can improve kidney region fibrosis by thing, be reduced in the expression of type i collagen and α-smooth muscle actin in one-sided urethral obstruction kidney.
As shown in Figure 2 B, PCR in real time testing result shows, after one-sided urethral obstruction the 7th day, and the expression of type i collagen and α-smooth muscle actin is to raise, but after input strikes the plasmid of low dynein light chain roadblock-2 type and suppress, it is just expressed and reduces.
As shown in Figure 2 C, Western blotting result shows, after one-sided urethral obstruction the 7th day, and the expression of type i collagen and α-smooth muscle actin is to raise, but with striking the expression that reduces its Men after the treatment of plasmid of low dynein light chain roadblock-2 type.
In summary, the Therapeutic Method of constrain dynein light chain roadblock-2 type effectively suppresses the activation of TGF-beta signal, and the phosphorylation mechanism of the suppressing Smad3 albumen activation of removing Smad3, and the progress of the interior renal fibrosis of minimizing body and termination renal fibrosis, suppress expression and α 2 (I) collagen promoter activity that dynein light chain-2 type has also reduced NTx.We also find, the expression of expression inhibiting α-smooth muscle actin in renal cells fibrotic processes of compacting dynein light chain-2 type, this may be to abolish the activation of autocrine TGF-beta signal path in renal fibrosis process because suppress dynein light chain-2 type.So the inhibition of dynein light chain roadblock-2 type will be a kind of effective treatment of hitting chronic kidney disease.
Above content is in conjunction with concrete/further description made for the present invention preferred embodiment, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention; without departing from the inventive concept of the premise; its embodiment that can also describe these is made some substituting or modification, and these substitute or variant all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. by suppressing the application of the material of dynein light chain roadblock-2 type for the preparation for the treatment of nephropathy diabetes medicament, it is characterized in that, use is struck low dynein light chain roadblock-2 type plasmid and is made described medicine.
2. application as claimed in claim 1, is characterized in that, strikes low dynein light chain roadblock-2 type plasmid and mix to make described medicine with microbubble contrast agent described in inciting somebody to action.
3. application as claimed in claim 2, is characterized in that, described microbubble contrast agent is sulfur hexafluoride microbubble contrast agent, preferably
contrast agent.
4. application as claimed in claim 2 or claim 3, is characterized in that, described in strike low dynein light chain roadblock-2 type plasmid and described microbubble contrast agent volume ratio be 1:1.
5. the application as described in claim 1 to 4 any one, it is characterized in that, the plasmid that structure strikes low dynein light chain roadblock-2 type is that a short hairpin RNA (shRNA) for dynein light chain roadblock-2 type messenger RNA is synthesized in carrier, to produce the bobby pin RNA of dynein light chain roadblock-2 type, wherein, the sequence of described short hairpin RNA is (GGA CAA CTC AAC AAC TGT TCA ATA T), preferably, described carrier is pSilencer4.1 carrier (Invitrogen, Carlsbad, VA, USA).
6. application as claimed in claim 5, is characterized in that, does not adopt and strike low dynein light chain roadblock-2 type plasmid described in endotoxic plasmid kit generation.
7. the application as described in claim 1 to 6 any one, is characterized in that, makes the intravenous injection of the microvesicle of the shRNA plasmid that strikes low dynein light chain roadblock-2 type that contains 1 μ M/ml.
8. a medicine for the treatment of nephropathy diabetes, is characterized in that, contains and strikes low dynein light chain roadblock-2 type plasmid.
9. medicine as claimed in claim 8, is characterized in that, also contains microbubble contrast agent, preferably, described in strike low dynein light chain roadblock-2 type plasmid and described microbubble contrast agent volume ratio be 1:1.
10. medicine as claimed in claim 8 or 9, is characterized in that, adopts the intravenous injection containing the shRNA plasmid that strikes low dynein light chain roadblock-2 type of 1 μ M/ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296488A (en) * | 2015-12-02 | 2016-02-03 | 中国人民解放军第三军医大学第三附属医院 | Application of ultrasound microbubble-mediated siRNA interference GRK4 in targeted adjustment of natriuresis and blood pressure level |
| CN105879064A (en) * | 2016-04-08 | 2016-08-24 | 江南大学 | Examination indexes of SonoVue ultrasonic contrast technology in diagnosis and treatment of tumor angiogenesis mimicry |
-
2014
- 2014-07-21 CN CN201410348885.4A patent/CN104189918A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| QUNYAN JIN，ET AL: "Requirement of a dynein light chain in TGFβ/Smad3 signaling", 《J CELL PHYSIOL》 * |
| RONG LI，ET AL: "The microRNA miR-433 promotes renal fibrosis by amplifying the TGF-β/Smad3-Azin1 pathway", 《KIDNEY INTERNATIONAL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296488A (en) * | 2015-12-02 | 2016-02-03 | 中国人民解放军第三军医大学第三附属医院 | Application of ultrasound microbubble-mediated siRNA interference GRK4 in targeted adjustment of natriuresis and blood pressure level |
| CN105879064A (en) * | 2016-04-08 | 2016-08-24 | 江南大学 | Examination indexes of SonoVue ultrasonic contrast technology in diagnosis and treatment of tumor angiogenesis mimicry |
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