Page 7 - JCTR-10-3

Xie et al. | Journal of Clinical and Translational Research 2024; 10(3): 180-190 181
supporting the idea that long-term gallstone stimulation can metabolic diseases, such as severe obesity, uncontrollable
lead to the transformation of benign hyperplasia into malignant hyperlipidemia, and diabetes; patients who took a large
gallbladder mucosal epithelial cells, leading to gallbladder dose of probiotics 3 months before the study; patients
cancer [7,8]. Recent studies have also reported that patients with who took somatostatin or other drugs affecting gallstone
asymptomatic gallstones have a significantly increased risk of formation, such as oral biliary acid therapy and proton-pump
the right-sided colon cancer after 15 years [9,10]. Therefore, inhibitors; pregnant women or long-term contraceptive
more emphasis should be placed on new prevention strategies users; patients who underwent endoscopic retrograde
against the formation of gallstones. cholangiopancreatography or intestinal surgery; and
The current understanding of the pathogenesis of gallstones patients who have primary sclerosis cholangitis, primary
is very complex, mainly involving local and systemic factors. biliary cholangitis, or Gilbert diseases.
The local factors include gallbladder wall motility disorder, local Based on the above selection criteria, we included 21 patients
persistent immune-mediated inflammation, mucin secretion who underwent gallstone surgery (gallstone group) and 20
and accumulation, cholesterol supersaturation, and solid crystal healthy patients without gallstones (control group). Clinical
precipitation [11,12]. Likewise, the systemic factors generally information on all patients was obtained, including gender,
include gene polymorphism, epigenetic factors, expression and age, body mass index (BMI), and cholesterol level. Gallstone,
activity of nuclear receptors, insulin resistance, slow intestinal bile, gallbladder mucosa, and feces specimens were collected
peristalsis, and increased cholesterol absorption [11,13]. The from the gallstone group, and feces were collected from the
activity of gut microbiota can dysregulate the lipids in bile and control group. This study was performed in accordance with
increase the excretion of bile acids, leading to the development the ethical standards of the responsible committee on human
of gallstones [14]. Some patients with gallstones suffer from experimentation (International Council for Medical Sciences)
discomforts such as belching, abdominal pain, abdominal and with the Helsinki Declaration. This study was approved
distension, and constipation for a long time. In recent years, much by the Institutional Research Ethics Committee of the Beijing
emphasis has been placed on understanding whether the formation Tiantan Hospital, Capital Medical University (KY 2020-032-02).
of gallstones is related to gut microbiota disorders. The screening All experimental personnel involved in specimen collection and
of biliary and intestinal microbiota has evolved from microscopic transportation received professional training.
characterization and identification to various culture technologies
(e.g., bacterial smear and culture methods) and molecular 2.2. Sample collection and processing
biology techniques. Nonetheless, the bacterial smear method During laparoscopic cholecystectomy, general surgeons
has limitations in identifying bacteria, while the bacterial culture collected the gallstones, bile (2 mL), and gallbladder mucosa
method has a low positive rate and limited efficacy in studying (about 1 × 1 cm in size). The assistant cleaned the surface of
biliary tract microorganisms. Conversely, molecular biology the gallstones and mucosa specimens with sterile normal saline
techniques (e.g., polymerase chain reaction [PCR] amplification, and stored them in sterile and labeled cryopreservation tubes.
fluorescence in situ hybridization, gene chip technology, 16S Fresh feces from the gallstone group were collected before
rRNA gene sequencing, and whole genome sequencing) have surgery, while feces from the control group were collected after
displayed advantages in accuracy, reliability, and repeatability in admission. Approximately 5 g of fresh feces were collected
studying the diversity and subtle changes of microbiota. Among and placed in sterile and labeled cryopreservation tubes for
them, 16S rRNA gene sequencing is the most suitable method for bacterial community detection. All specimens from the study
bacterial phylogeny and species classification. participants were placed in liquid nitrogen for at least 3 min for
In this study, the V3-V4 variable regions of the 16S rRNA
gene were sequenced to reveal the diversity of biliary and rapid freezing and then stored in a freezer at −80°C.
gut microbiota in patients with biliary stones. We aimed to 2.3. DNA extraction and bacterial 16S rRNA amplification and
investigate the alterations of biliary and gut microbiota and their sequencing
possible correlation to gallstone formation.
DNA was extracted from samples (0.5 g) using the QIAamp
2. Methods PowerFecal DNA Kit (QIAGEN, Germany) according to the
manufacturer’s protocols. Subsequently, the V3-V4 regions
2.1. Study design
of the bacterial 16S rDNA gene were amplified by PCR using
We recruited patients who underwent a physical examination primers 16s-336F (5’-GTACTCCTACGGGAGGCAGCA-3’)
at the Beijing Tiantan Hospital affiliated with the Capital and 16s-806R (5’-GTGGACTACHVGGGTWTCTAAT-3’).
Medical University from November 2019 to November 2020 The first PCR reaction was performed in a 25 μL mixture
based on the following inclusion and exclusion criteria: containing 5 μL 5× GC Buffer, 0.5 μL KAPA dNTP Mix, 0.5 μL
i. Inclusion criterion: Patients with gallstones that were KAPA HiFi HotStart DNA polymerase (KAPA Biosystems,
confirmed by abdominal B-mode ultrasound or computed America), 0.5 μL forward primer (10 pM), 0.5 μL reverse
tomography. primer (10 pM), and 50 – 100 ng of template DNA. PCR cycling
ii. Exclusion criteria: Patients who received antibiotics included 95°C denaturation for 3 min, followed by 25 cycles at
within the past 3 months; the presence of other serious 95°C for 30 s, 55°C annealing for 30 s, and 72°C elongation

DOI: https://doi.org/10.36922/jctr.23.00118