Medical application of GPD1L in aortic aneurysm treatment

Abstract

The invention provides medical application of GPD1L in treatment of aortic aneurysm. Comprising application of a substance for overexpressing the GPD1L gene or improving the GPD1L protein expression quantity in preparation of drugs for preventing and / or treating aortic aneurysm. The substance is a lentiviral vector of GPD1L. The lentiviral vector of the GPD1L is constructed by the following steps: connecting a GPD1L gene sequence as shown in SEQ ID NO.1 between SpeI and BamHI restriction enzyme cutting sites of a lentiviral vector pLVX-FLEX-EF1a-ZsGreen, so as to obtain the lentiviral vector of the GPD1L. The substance is a lentiviral vector transfection system of GPD1L. The lentiviral vector transfection system is constructed by transfecting an HEK293 cell with a lentiviral vector. The substance overexpresses a GPD1L gene in smooth muscle cells or improves the expression quantity of GPD1L protein. The GPD1L protein in the smooth muscle cell is specifically overexpressed, so that the morbidity of the aortic aneurysm and the vasculopathy can be reduced, a new target spot is provided for treating the aortic aneurysm, and the GPD1L protein has important clinical significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the use of glycerol-3-phosphate dehydrogenase 1 like protein (GPD1L) in the treatment of aortic aneurysm. BACKGROUND

[0002] Aortic aneurysm is a serious cardiovascular disease that seriously endangers human health, which is manifested as irreversible aneurysmal dilatation of the aortic vessel wall. The rupture of the aneurysm can cause massive bleeding in the abdominal cavity and lead to patient death. Due to the rising incidence of aortic aneurysm risk factors such as hypertension, atherosclerosis, the acceleration of population aging, and the bad living habit of smoking, the incidence of aortic aneurysm is showing an upward trend in the population. Global statistics show that the number of deaths due to aortic aneurysm rupture reaches 150,000 to 200,000 per year.

[0003] At present, there is a lack of effective prevention and treatment drugs in clinical practice, and the treatment methods for aortic aneurysm are mainly surgical operation or interventional therapy, but there are problems of high difficulty, high cost and high incidence of complications. Some patients cannot undergo surgery due to complications or age, and some patients will relapse after treatment and need surgical intervention again.

[0004] The non-surgical treatment method for aortic aneurysm in clinical practice is mainly to reduce the risk of combined cardiovascular diseases by smoking cessation, blood pressure control and blood lipid regulation, so as to slow down the growth rate of aneurysm, and there is no effective drug for preventing and treating aortic aneurysm. Therefore, it is of great significance to explore the molecular mechanism of aortic aneurysm, find the key intervention target, and early diagnosis, prevention and drug treatment.

[0005] GPD1L (glycerol-3-phosphate dehydrogenase 1 like) is the full name of glycerol-3-phosphate dehydrogenase 1 like protein, which is expressed in vascular smooth muscle cells. GPD1L has enzyme catalytic activity, which catalyzes the conversion of phospho-dihydroxyacetone, an intermediate product in glycolysis, to glycerol-3-phosphate, and converts NADH in the cytoplasm to NAD + , which helps to transport the NADH reduction equivalent (H) produced by glycolysis to mitochondria, supports oxidative phosphorylation and ATP generation, and participates in cellular energy metabolism.

[0006] Current research shows that GPD1L plays an important role in a variety of tumors and arrhythmias, but its role in aortic aneurysm has not been reported. SUMMARY

[0007] To solve the above technical problems in the prior art, the present application provides the medical use of GPD1L in the treatment of aortic aneurysm.

[0008] The technical scheme of the present application is:

[0009] The application provides application of a substance overexpressing a GPD1L gene or increasing expression of GPD1L protein in preparation of a medicine for preventing and / or treating aortic aneurysm.

[0010] The nucleotide sequence of the GPD1L gene is shown in SEQ ID NO. 1:

[0011] The amino acid sequence of the GPD1L protein is shown as SEQ ID NO. 2: MAAAPLKVCIVGSGNWGSAVAKIIGSNVKTLQKFSSTVKMWVFEETVNGRKLTDIINNDHENVKYLPGHKLPENVVAVPNLSEAVQDADLLVFVIPHQFIHKICDEITGRVPEKALGITLIKGIDEGPDGLKLISDIIREKMGIDISVLMGANIASEVAAEKFCETTIGSKVMQNGLLFKELLQTPNFRITVVDDADTVELCGALKNIVAVGAGFCDGLRCGDNTKAAVIRLGLMEMIAFAKIFCKGQVSTATFLESCGVADLITTCYGGRNRRVAEAFARTGKTIEELEKELLNGQKLQGPQTSAEVYRILRQKGLLDKFPLFTAVYQICYEGRPVTQMLSCLQSHPEHI.

[0012] Further, the substance is a lentivirus vector of GPD1L.

[0013] Further, the lentivirus vector of GPD1L is constructed by the following steps: connecting the GPD1L gene sequence shown in SEQ ID NO. 1 into the SpeI and BamHI enzyme cutting sites of the lentivirus vector pLVX-FLEX-EF1a-ZsGreen.

[0014] Further, the nucleotide sequence of the lentivirus vector of GPD1L is shown as SEQ ID NO. 3.

[0015] Further, the substance is a lentivirus vector transfection system of GPD1L.

[0016] Further, the lentivirus vector transfection system is constructed by transfecting the lentivirus vector of GPD1L into HEK293 cells.

[0017] Further, the substance overexpresses the GPD1L gene or increases the expression amount of the GPD1L protein in smooth muscle cells.

[0018] The beneficial effects of the present application are: Vascular smooth muscle cells (VSMC) are the main cell type to maintain the integrity of the vascular wall and vascular tension, and the dysfunction of VSMC is an important pathological feature of aortic aneurysm. In the course of aortic aneurysm, the number of VSMC cells decreases, which causes the gradual degeneration and thinning of the media of the vascular wall, the decrease of the elasticity of the vascular wall and the increase of pathological dilation, and finally leads to vascular rupture. The present application finds that GPD1L can participate in the progression of aortic aneurysm by regulating the functional homeostasis of VSMC, thereby better treating aortic aneurysm and greatly improving the quality of life of patients with aortic aneurysm.

[0019] The present application can reduce the incidence of aortic aneurysm and alleviate vascular lesions by specifically overexpressing GPD1L protein in smooth muscle cells, thereby providing a new target for treating aortic aneurysm and having important clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 8-week-old ApoE - / - Male mice were implanted with a micro-osmotic pump containing angiotensin II (Ang II, 1000 ng / kg / min) subcutaneously on the back for 4 weeks to construct an aortic aneurysm model, and the vascular tissue of the mice was extracted. The expression level of GPD1L in the blood vessels of the mice was detected by Western Blot.

[0021] Figure 2 6-week-old ApoE - / - / Tagln Cre Male mice were injected with a control empty lentivirus (Lenti-Vector) or a GPD1L lentivirus (Lenti-GPD1L) via the tail vein. The lentivirus plasmid vector contains two inverted LoxP, and the Cre recombinase in the smooth muscle cells induces the inversion of the GPD1L cDNA sequence between the loxP, thereby inducing the specific overexpression of GPD1L in the smooth muscle cells. After 2 weeks, a micro-osmotic pump containing saline (Saline) or Ang II (1000 ng / kg / min) was implanted subcutaneously, and the model was constructed for 4 weeks to construct an aortic aneurysm model, and the formation of aortic aneurysm was observed. Among them: A: The general diagram of the aorta of the mice collected after modeling for 4 weeks, and the scale is 2 mm; B: Quantification of the maximum diameter of the aorta; C: Detection of the incidence of aortic aneurysm in mice.

[0022] Figure 3 The collected vascular tissue of the experimental mice was cut to obtain a segment of the suprarenal aorta (tumor area) for polyformaldehyde fixation, paraffin embedding and sectioning, and staining; wherein: A: Representative diagram of HE tissue staining, the magnified scale is 50 μm, and the scale in the upper right corner is 400 μm; B: Representative image of EVG tissue staining, scale bar 50 pm, scale bar in the upper right corner 400 pm.

[0023] Figure 4 Lentiviral vector map. DETAILED DESCRIPTION

[0024] The application will be further explained in connection with the following examples, which do not limit the application in any way. Example 1

[0025] To explore the changes of GPD1L in aortic aneurysm, we selected 8-week-old Apoe - / - Tagln cre Male mice were implanted with a micro-osmotic pump containing angiotensin II (Ang II, 1000 ng / kg / min) subcutaneously for 4 weeks to construct an aortic aneurysm animal model. The mouse diseased vascular tissue was extracted, and the expression level of GPD1L in the mouse blood vessels was detected by Western Blot.

[0026] The Western Blot procedure is as follows: (1) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separating gel and 3% concentrated gel. The preparation method of 12% separating gel is to mix 1.6 mL of deionized water, 2.0 mL of 30% AA mother liquor, 1.3 mL of 1.5M Tris-HCl (pH 8.8), 50 μL of 10% SDS, 50 μL of 10% ammonium persulfate (APS), and 5 μL of TEMED, with a total volume of 5 mL. After 30 min of room temperature solidification, prepare 3% concentrated gel. The preparation method of 3% concentrated gel is to mix 1.8 mL of deionized water, 0.3 mL of 30% AA mother liquor, 0.75 mL of 1M Tris-HCl (pH 6.8), 30 μL of 10% SDS, 50 μL of 10% ammonium persulfate (APS), and 5 μL of TEMED, with a total volume of 3 mL. After 30 min of room temperature solidification, use it.

[0027] Take 15 μL of sample and 3 μL of loading buffer, mix well. After boiling for 5 min to denature the protein, load about 30 μg per well. Electrophorese at 110 V constant voltage for about 90 min until the bromophenol blue disappears.

[0028] (2) Transferring membrane: after the electrophoresis, cut the concentrated gel, and immerse the gel in the protein transfer buffer (3.6 g / L Tris, 200 ml / L methanol, 17.3 g / L glycine) for 10-20 min. The wet transfer method is used to transfer the protein band to the PVDF membrane (the SDS-gel is located at the negative electrode, and the PVDF membrane is located at the positive electrode), and the constant current electrophoresis is performed at 0.3 A for 80 min.

[0029] (3) Blocking: after the transferring membrane is completed, the PVDF membrane is taken out, soaked in PBS for 5 min, and then immersed in the TBST containing 5% skimmed milk powder for 1 h at room temperature.

[0030] (4) Primary antibody incubation: after the blocking is completed, the membrane is placed in the hybridization bag, the antibody is added, and the shaking incubation is performed at 4°C overnight.

[0031] (5) Secondary antibody binding: the membrane is washed with TBST for 10 min for 3 times. The antibody of the corresponding species of the primary antibody is added, and the incubation is performed at room temperature for 1 h. The membrane is washed with TBST for 10 min for 3 times.

[0032] (6) ECL color development: the ECL color development liquid A and B are mixed immediately before use, and are uniformly added to the surface of the membrane. The film is exposed to light, and the results are observed.

[0033] The experimental results show that, compared with the control group, the expression level of GPD1L in the mouse vascular tissue of the aorta decreases Figure 1 ).

[0034] The above results show that GPD1L may play an important role in the progression of the aortic aneurysm. Example 2

[0035] In order to further explore the role of GPD1L in the aortic aneurysm, 6-week-old ApoE - / - Tagln cre The control empty lentivirus (Lenti-Vector) or GPD1L lentivirus (Lenti-GPD1L) is injected into the tail vein of the mouse. The lentivirus plasmid vector contains two reverse LoxP. The Cre recombinase in the smooth muscle cell induces the Gpd1l cDNA sequence between the loxP to be flipped, so as to induce the specific overexpression of Gpd1l in the smooth muscle cell (the GPD1L used in the embodiment of the application is the mouse GPD1L gene, and the mGPD1L is the sequence shown in SEQ ID NO. 1). After 2 weeks, the physiological saline (Saline) / angiotensin II (Ang II) micro-osmotic pump is implanted for 4 weeks. The blood vessel diameter of the mouse is monitored by ultrasonic Doppler during the research period. After 4 weeks, the mouse aortic blood vessels are separated, and the aortic blood vessel general picture is taken to observe the formation of the aortic aneurysm.

[0036] The specific experimental steps are as follows: (1) Take ApoE - / - Tagln cre Male mice, 6-week-old mice were injected with control empty lentivirus and GPD1L lentivirus (Lenti-Vector / Lenti-GPD1L titer 10 9 ifu / each) via tail vein injection, respectively.

[0037] (2) The mice were bred in SPF level feeding environment for 2 weeks to make the lentivirus stably expressed in smooth muscle cells.

[0038] (3) When the mice were 8 weeks old, Ang II micro-osmotic pumps were implanted at 1000 ng / min / kg, and the control group was given normal saline.

[0039] (4) The Ang II micro-osmotic pump was implanted for 4 weeks, and the blood vessel diameter of the mice was monitored by ultrasound Doppler during the period. After 4 weeks, the aortic blood vessel tissue was extracted, and the related pathological indicators were measured.

[0040] (5) The GPD1L lentivirus was synthesized by Wuhan Winoseq Biotechnology Co., Ltd.

[0041] The specific lentivirus construction method is as follows: (1) Control empty lentivirus (Lenti-Vector), i.e. lentivirus vector pLVX-FLEX-EF1a-ZsGreen (manufacturer: Wuhan Winoseq Biotechnology Co., Ltd., recorded in J Clin Invest, 2025, 135(11)).

[0042] (2) The construction method of GPD1L lentivirus (Lenti-GPD1L) is as follows: the mGPD1L gene template with the nucleotide sequence shown in SEQ ID NO. 1 is synthesized, which is constructed into the SpeI and BamHI enzyme digestion sites of the lentivirus vector pLVX-FLEX-EF1a-ZsGreen, and then the enzyme digestion and sequencing identification are performed, and the obtained lentivirus is packaged by transfecting HEK293 cells, and the obtained lentivirus is amplified, purified and the titer is determined (Lenti-Vector / Lenti-GPD1L titer 10 Figure 4 ), and the specific steps are as follows: ① to ③.

[0043] In this example, the primers used are: CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3' (SEQ ID NO. 4).

[0044] The plasmid construction process is as follows: ① The plasmid vector pLVX-FLEX-EF1a-ZsGreen (manufacturer: Wuhan Winoseq Biotechnology Co., Ltd.) is digested with SpeI and BamHI, and the digested product is connected with the mGPD1L gene template to construct the lentivirus vector pLVX-FLEX-EF1a-ZsGreen-mGPD1L. SpeI and BamHIDouble enzyme digestion: enzyme digestion reaction in 37℃ water bath reaction for 3 h, and the enzyme digestion system is as follows: 2 μg of pLVX-FLEX-EF1a-ZsGreen, 3 μl of 10×Buffer CutSmart, 1 μl of SpeI and 1 μl of BamHI, Add deionized water to make up the volume to 30 μl.

[0045] 1% agarose gel electrophoresis to recover the plasmid vector pLVX-FLEX-EF1a-ZsGreen SpeI+BamHI enzyme digestion large fragment.

[0046] The construction of the target gene plasmid (pUC57-mGpd1l) is to insert the mGPD1L gene sequence between the SpeI / BamHI enzyme digestion sites of the pUC57 vector, and the specific steps are as follows: mGpd1l synthetic sequence size: 1140 bp; enzyme digestion site: SpeI-BamHI; vector: pUC57.

[0047] The above pUC57-mGpd1l SpeI and BamHI Double enzyme digestion, enzyme digestion reaction in 37℃ water bath reaction for 3 h, and the enzyme digestion system is as follows: 25 μl of pUC57-mGpd1l, 3 μl of 10×Buffer CutSmart, 1 μl of SpeI and 1 μl of BamHI .

[0048] 1% agarose gel electrophoresis to recover the plasmid pUC57-mGpd1l SpeI+BamHI enzyme digestion small fragment, i.e. the sequence shown in SEQ ID NO. 5: ACTAGTGGATCC .

[0049] Underlined ACTAGT is SpeI restriction enzyme recognition site, GGATCC is BamHI restriction enzyme recognition site, and the bolded part is 3xFLAG protein fusion tag sequence.

[0050] ③Step 1 pLVX-FLEX-EF1a-ZsGreen SpeI+BamHI enzyme cutting large fragment was connected with step 2 pUC57-mGpd1l SpeI+BamHI enzyme cutting small fragment, the connection reaction was reacted at 22℃ for 3 h, and the reaction system was as follows: 6 μl of mGpd1l fragment, 2 μl of pLVX-FLEX-EF1a-ZsGreen large fragment, 1 μl of 10xLigase Buffer and 1 μl of T4 DNA Ligase.

[0051] ④Connection product transformation: 10 μl of connection product was mixed with 100 μl of JM109 competent bacteria, then ice bath for 30 min, 42℃ heat shock for 60 s, immediately placed on ice for 2 min, 400 μl of preheated to room temperature LB medium was added, 37℃ constant temperature incubator was cultured for 1 h, 4000 rpm centrifuged for 3 min, 400 μl of culture supernatant was discarded, the remaining 100 μl was mixed with pipette and evenly coated on LB plate containing 100 μg / ml ampicillin resistance, 37℃ constant temperature incubator was cultured overnight.

[0052] ⑤Three single colonies were picked and inoculated in 5 ml of LB culture containing 100 μg / ml ampicillin resistance, 250 rpm, 37℃ constant temperature incubator was cultured overnight, small amount of plasmid extraction kit was used to extract plasmid, the plasmid was digested with SpeI+BamHI for enzyme digestion identification, and the correct clone was picked for sequencing verification.

[0053] The sequencing result was consistent with the design, and the specific vector full sequence was as shown in SEQ ID NO. 3:

[0054] Results show that compared with the physiological saline group, the aortic aneurysm mice with Lenti-Vector + Ang II implanted pump, the aorta of the mice showed obvious pathological dilation, the aortic diameter increased significantly, the incidence of abdominal aortic aneurysm and the mortality increased significantly. Compared with the Lenti-Vector + Ang II implanted pump group, the aortic aneurysm mice in the Lenti-GPD1L + Ang II implanted pump group, the aortic diameter expansion and the incidence of abdominal aortic aneurysm were inhibited. Figure 2 ).

[0055] The collected experimental mouse vascular tissues were cut to obtain a segment of the supra-renal aorta (tumor area) for polyformaldehyde fixation, paraffin embedding and sectioning, and then HE and EVG staining was performed. The results showed that overexpression of GPD1L can significantly improve the abnormal expansion of mouse aorta caused by Ang II implanted pump and the disorder and fracture of elastic fibers. Figure 3 ).

[0056] The above results further prove that the increase of GPD1L expression can reduce the mortality of abdominal aortic aneurysm mice, delay the progression of aortic aneurysm disease, and thus improve the survival rate of mice.

[0057] The above only describes the preferred embodiments of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of a substance overexpressing a GPD1L gene or increasing the expression amount of a GPD1L protein in the preparation of a drug for preventing and / or treating aortic aneurysm.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the GPD1L gene is shown as SEQ ID NO.

1.

3. Use according to claim 1, characterized in that, The amino acid sequence of the GPD1L protein is shown as SEQ ID NO.

2.

4. Use according to claim 3, characterized in that, The substance is a lentiviral vector of GPD1L.

5. Use according to claim 3, characterized in that, The lentiviral vector of GPD1L is constructed by connecting the GPD1L gene sequence of SEQ ID NO. 1 into the SpeI and BamHI enzyme cutting sites of the lentiviral vector pLVX-FLEX-EF1a-ZsGreen.

6. Use according to claim 5, characterized in that, The nucleotide sequence of the lentiviral vector of GPD1L is shown as SEQ ID NO.

3.

7. Use according to claim 1, characterized in that, The substance is a lentiviral vector transfection system of GPD1L.

8. Use according to claim 7, characterized in that, The lentiviral vector transfection system is constructed by transfecting the lentiviral vector of GPD1L into HEK293 cells.

9. The use according to claim 1, characterized in that, The substance overexpresses a GPD1L gene or increases the expression amount of a GPD1L protein in smooth muscle cells.

Images