Observational Study of the Correlation Between Peripheral Treg Cell Senescence and Serum Total Cholesterol Level
Conditions: Hypercholesterolemia
Sponsor: Tianjin Medical University

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 1 - 
 
 
 
 
 
 
 
 
 
 
 
 
 
Observational study of the correlation between 
peripheral Treg cell senescence  
and serum total cholesterol level 
 
 
 
 
 
 
 
 
 
 
 
 
Version: 1.0 
 
Date: December 1st, 2022 
 
 
 
 
Ethics Committee 
 
Tianjin Medical University General hospital 

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 2 - 
Protocol Abstract 
Title 
Observational study of the correlation between peripheral Treg cell 
senescence and serum total cholesterol level 
Ethics 
Committee 
(Grant Number) 
The ethics committee of Tianjin Medical University General Hospital 
(IRB2022-YX-244-01) 
Principal Investigator
Ping Lei (Ph.D.) 
Sponsor 
Tianjin Medical University General Hospital 
Fundings 
Natural Science Foundation of China (Grant No. 82071394, 82072166)
Execute Time 
12/01/2022-03/31/2023 
Recruiting Time 
01/01/2023-03/31/2023 
Objective 
Clarify the correlation between human peripheral Treg cell senescence 
and serum total cholesterol level 
Study Type 
Observational 
Time Perspective 
Cross-Sectional 
Enrollment 
200 participants 
Biospecimen 
Description: Whole blood and serum 
Retention: Samples with DNA 
Sampling Method 
Probability sample 
Condition 
Hypercholesterolemia 
Inclusion Criteria 
1) Age ≥ 30 and ≤ 60 years old with independent behavior ability. 
2) The participants need to fully understand the purpose and content of 
the study, and voluntarily participate in the study and sign the informed 
consent. 
Exclusion Criteria 
1) Pregnant or lactating women. 
2) Acute and severe diseases in the last 3 months, including but not 
limited to, acute myocardial infarction, acute cerebral infarction, 
cerebral hemorrhage, circulatory failure, respiratory failure (internal 
diseases), trauma requiring hospitalization, or undergoes surgery under 
general anesthesia (surgical diseases). 

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 3 - 
3) History of severe diseases, including but not limited to, tumors, 
serious hematological diseases, serious cardiopulmonary diseases 
(interventional therapy for coronary artery disease, atrial fibrillation, 
chronic obstructive pulmonary disease, etc.), renal failure, liver failure, 
old stroke with serious sequelae. 
4) Have participated in clinical trials in the past 3 months. 
5) The investigator considers that not appropriate for inclusion. 
Outcome Measures 
Treg cell senescence evaluated by the beta-galactosidase (SA-betagal) 
activity test. 
Reference standard 
Protocols 
to 
detect 
senescence-associated 
beta-galactosidase 
(SA-betagal) activity, a biomarker of senescent cells in culture and in 
vivo. Nat Protoc 4, 1798-1806, doi:10.1038/nprot.2009.191 (2009) 
Statistical Analysis 
All statistical analyses were performed using SPSS Statistics Version 
27.0 (IBM, Armonk, NY, USA). A two-tailed p value of less than 0.05 
was considered to be statistically significant. 

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 4 - 
1. Research Background and Objectives 
Hypercholesterolemia is a risk factor for the occurrence and development of atherosclerosis. 
Recent animal studies have found that increased serum cholesterol level is associated with peripheral 
Treg cell senescence, but clinical evidence is still lacking. The purpose of this study is to analyze the 
correlation between human peripheral Treg cell senescence and serum total cholesterol level using 
clinical blood samples, thus laying a foundation for the establishment of novel therapeutic strategies 
for atherosclerosis based on the regulation of Treg cell senescence. 
2. Study design 
2.1 Overall design 
This research is an observational cross-sectional study. 
2.2 Estimated number of subjects 
Estimated by statistical analysis, a total of 200 subjects were randomly selected from the 
population receiving physical examination in the Cadre Physical examination Center of Tianjin 
Medical University General Hospital from January 2023 to March 2023. 
3. Inclusion, Exclusion and Withdrawal Criteria 
3.1 Inclusion Criteria 
1) Age ≥ 30 and ≤ 60 years old with independent behavior ability. 
2) The participants need to fully understand the purpose and content of the study, and 
voluntarily participate in the study and sign the informed consent. 
3.2 Exclusion Criteria 
1) Pregnant or lactating women. 
2) Acute and severe diseases in the last 3 months, including but not limited to, acute 
myocardial infarction, acute cerebral infarction, cerebral hemorrhage, circulatory failure, respiratory 
failure (internal diseases), trauma requiring hospitalization, or undergoes surgery under general 
anesthesia (surgical diseases). 
3) History of severe diseases, including but not limited to, tumors, serious hematological 
diseases, serious cardiopulmonary diseases (interventional therapy for coronary artery disease, atrial 
fibrillation, chronic obstructive pulmonary disease, etc.), renal failure, liver failure, old stroke with 
serious sequelae. 
4) Have participated in clinical trials in the past 3 months. 

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 5 - 
5) The investigator considers that not appropriate for inclusion. 
3.3 Withdrawal criteria 
1) The subject decides to withdraw from the study. 
2) The doctor or the researcher asks the subject to withdraw from the study (e.g. poor 
compliance). 
4. Study procedure 
Researchers initiate the study according to the inclusion/exclusion criteria. After confirming 
the subject has signed the informed consent, doctors will record the past medical history and 
medication (especially hypolipidemic drugs) in the past 3 months. Thereafter, an additional 6 ml of 
venous blood will be drawn during the routine blood tests for examinations of Treg cell senescence. 
5. Evaluation Parameters 
Treg cell senescence will be evaluated by the beta-galactosidase (SA-betagal) activity test. 
6. Withdrawal of the subject 
The subject has the right to withdraw from this trial at any time for any reason. The researcher 
should have a thorough understanding and a record of the reasons for withdrawal. Doctors and 
researchers also have the right to stop the subject from continuing to participate in the trial at any 
time during the research. Besides, subjects with early withdrawal should not be replaced by others. 
As too many subjects withdrawing from the trial will lead to unreliable results, unnecessary 
withdrawal should be avoided. The falling rate of the trial should be controlled within 20%. 
7. Statistical analysis 
1. Pearson correlation coefficient was used to evaluate the correlation between the number of Treg 
cells in peripheral blood and serum total cholesterol level. 
2. Taking the serum total cholesterol level of 5.17 mmol/L as the cut-off value, the subjects were 
divided into normal cholesterol level group and hypercholesterolemia group. Subsequently, the 
Propensity Score Matching (PSM) method was used to match the subjects according to gender, age 
and past medical history, and the Student's t-test was performed to compare the difference of Treg 
cell senescence between the two groups. 
3. The serum total cholesterol level was stratified (e.g., trisection or quartering), and the difference 
of Treg cell senescence among all groups was compared using one-way ANOVA followed by 
LSD post-hoc test. 

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 6 - 
8. Trial Management 
8.1 Ethics 
To obtain the approval documents of the clinical trial, researchers should submit the trial 
protocol and a copy of the research documents including the informed consent to the ethics 
committee. 
The approval documents from the ethics committee should be accompanied by the name list of 
ethics committee members and their respective responsibilities. These documents will be delivered 
to the researchers in written form before the start of the study.  
Any safety-related issues must be promptly reported to the ethics committee, which includes 
revision on the trial protocol and modification on the subject information page. The end or early 
termination of the trial should also be reported. 
8.2 Informed consent and data protection agreement 
It is the responsibility of the researchers to explain the objectives, study procedures, benefits 
and potential risks of the trial for each subject. The researchers should receive the informed consent 
signed by the subject before starting the trial, and keep it properly. The subject should also permit 
researchers and the clinical trial management agency to check his or her original data. Thus, the 
reliability of the research findings could be ensured. 
The personal information of each subject, including name, gender, age, home address and 
telephone number, should be collected in detail. And the researcher/doctor should give the subject 
his or her contact information, so that the subject can contact with him or her when needed. This is 
also helpful for the research and medical care. 
8.3 Subjects privacy 
The researchers should protect the privacy of the subject. All research documents can only be 
identified using the subject's number instead of name or physical examination number. In addition, 
the grouped table that records the correspondence between the subject's name and number can only 
be kept by the researcher, and could not be submitted to any institution or individual. 
8.4 Modification of the trial protocol 
The trial protocol is approved by the ethics committee before starting the trial. During the 
research, any proposed modification on the trial protocol should be reported to the principal 
investigator, and then be submitted to the ethics committee for review. The clinical research can 
only be resumed after the re-approval of the ethics committee. 

Peripheral Treg cell senescence & serum total cholesterol level                      Identifier:  
 
Tianjin Medical University General Hospital                                                         - 7 - 
8.5 Original records certification 
Researchers should ensure that the subject's privacy is protected when collecting and 
organizing data. In addition, the data manager is authorized to review the original records in order 
to confirm the accuracy of the original data. 
8.6 Documents on file 
According to relevant laws and regulations, researchers should keep the original records 
properly for at least 5 years from the end of the study. 
8.7 Quality control 
The clinical trial management agency has the authority to review the study progress, in order to 
ensure that the trial is carried out as predetermined and that the research data be veritably recorded.