醫學院 109 學年度學生暑期研究

探討 RAS 抑制劑與影響 ACE2 基因表現程度之研究

An investigation of the candidate RAS inhibitors on the expression of ACE2 genes

一、 指導老師(中文姓名/英文姓名) 劉秉彥醫師 Dr. Pin-Yen Liu

二、 學生(系所/年級/中文姓名/英文姓名) 醫學系二年級 蔡如庭 Ru-Ting Tsai
學生聯絡電話:0952197581
學生 email: tiffany900505@gmail.com
三、 研究背景

Angiotensinogen
Renin
ACE-2
Ang I Ang 1-9

ACE ACE
ACE-2
Ang II Ang 1-7 Ang 1-5

ACE

AT2R AT1R masR

ACE (angiotensin-converting enzyme)-2 is a component of the renin-angiotensin system

(RAS). Accumulating evidence shows that ACE-2 provides protective effects in peripheral tissues
and has great potential for the treatment of RAS-related diseases. The emerging concept is that an
imbalance in ACE-2/Ang-(1-7) and ACE/Ang-II axes is critical in the development of
cardiovascular diseases. If ACE-2/Ang-(1-7) axe is blocked, the Ang-II is unable to be broken down.
The overexpressed Ang-II will bind to AT1R receptor and lead to its over-stimulation. Under the
circumstances, the chances of vasoconstriction, hypertrophy and atherosclerosis are getting higher.
Besides, the central role of ACE-2, therefore, appears to counter ACE activity by reducing Ang-II
bioavailability and increasing Ang-(1-7) formation.

As for ACE, which is also a component of the RAS, converts hormone Ang-I to active
vasoconstrictor Ang-II and indirectly increases blood pressure by constricting blood vessels.
Therefore, ACE inhibitors are widely used as pharmaceutical drugs for treatment of cardiovascular
diseases.

https://www.ahajournals.org/doi/full/10.1161/circresaha.113.301271
 There are several drugs serving as ACE inhibitors, such as ACEI and AT1R (also known as
ARB). What we would like to know is whether the dosages we apply will affect the ACE-2 gene
expression or not, and which one will lead to the greatest influence on this system.

四、 研究方法
The experimental procedures are as follows:
1. Cell culture
ACE-2 expressed abundantly in the epithelial cell system. We will use HEK293T, a human
kidney epithelial cell line, supplemented with DMEM with 10% FBS and 1% P/S, and then
being incubated in a humidified atmosphere at 37℃ with 5% CO2.
2. ACE-2 plasmid (obtained from commercial vendor)
(1) Name: pCMV3-hACE2-GFPSpark
(2) Length: 6848 base pairs (b.p.) (pCMV3) + 2418 b.p. (hACE2-GFPSpark) = 9266 b.p.
3. Transfection
When 80% confluency, cells will be transfected with ACE-2 plasmid using Lipofectamine 2000.
Four hours after transfection, cells will be incubated with regular medium for 24 hours and
collected after fluorescence microscopy imaging.
4. Drug treatments to cells
(1) Ramipril (ACE inhibitor) (3) Spironolactone (Diuretic)
(2) Valsartan (AT1R/ARB) (4) Entresto (ARB + NEPI)
All drugs used in this study are obtained from commercial vendors. Cells will be starved in
serum-free medium for 24 hours prior to the addition of 1-10 μM drugs for 24 hours. MTT
assay will be conducted initially.
5. Fluorescence microscopy
Transfected cells will be observed with Olympus IX71 inverted fluorescence microscopy.
6. Cell lysis and Western Blot
To obtain whole-cell extracts, cells will be washed with icy PBS, and lysed in RIPA lysis buffer
supplemented with protease inhibitor cocktail. After scraped from cell plate, cell extracts will be
cleared by centrifugation at 4℃. Cell extracts will be quantified using BCA assay, and equal
amounts of proteins will be mixed with SDS sample buffer, boiled, separated by SDS-PAGE,
and blotted onto PVDF membrane. Membrane will be blocked with 5% milk for 1 hour and
incubated at 4℃ for overnight with ACE-2 primary antibody diluted in TBST containing 5%
BSA. After washing, membrane will be incubated with HRP-conjugated anti-rabbit IgG, and the
protein bands will be revealed by ECL system.

五、 預期成果
Through the experimental treatment, the expected results will be as follows:
1. The dosages will affect the gene expression of ACE-2 in HEK293T cells.
2. By observing the distribution of GFP on ACE-2 under fluorescence microscope and the results
from Western Blot, we can distinguish the different outcomes between various treatments.