PCR-DGGE를 통해 분석한 항암치료에 따른 장내 미생물 변화 A PCR Denaturing Gradient Gel Electrophoresis (DGGE) Analysis of Intestinal Microbiota in Gastric Cancer Patients Taking Anticancer Agents원문보기
인체의 장내에 존재하는 장내 미생물은 서로 공생 또는 길항 관계를 유지하며 우리 몸의 면역 방어 기전에 중요한 요소로 작용한다. 본 연구는 항암제가 위암 환자의 장내 미생물 생태계에 미치는 영향을 조사 하였다. 항암치료를 받는 환자의 분변에서 genomic DNA를 추출하고, 16S rDNA 유전자에 대한 denaturing gradient gel electrophoresis (DGGE)를 수행하였다. 분석된 균주는 개체간의 차이가 있었으나, 대부분 사람의 장내에 살고 있는 normal flora로 동정되었다. 모든 분변에 존재하는 5 개 밴드의 서열 분석 결과에 의하면 Faecalibacterium prausnitzii, Morganella morganii 및 Uncultured bacterium sp.가 나타났고, 항암제 처리 후 Sphingomonas paucimobilis, Lactobacillus gasseri, Parabacteroides distasonis 및 Enterobacter sp.가 증가하였다. 이 연구에서 probiotic으로 알려진 Bifidobacterium과 Lactobacillus를 특이적 PCR primer를 이용하여 동정한 결과, 항암제 투여로 인해 Bifidobacterium과 Lactobacillus의 개체군이 현저하게 줄어들어 diarrhea와 같은 부작용의 원인을 예상하게 하며, 장내 생태계의 주요 박테리아 집단에도 중요한 영향을 미치는 것을 알 수 있었다. 이러한 결과는 항암제 투여와 같이 시간의 흐름에 따른 균총의 변화를 시각적으로 모니터링하기 위하여 PCR-DGGE분석법이 유용하다는 것을 나타낸다.
인체의 장내에 존재하는 장내 미생물은 서로 공생 또는 길항 관계를 유지하며 우리 몸의 면역 방어 기전에 중요한 요소로 작용한다. 본 연구는 항암제가 위암 환자의 장내 미생물 생태계에 미치는 영향을 조사 하였다. 항암치료를 받는 환자의 분변에서 genomic DNA를 추출하고, 16S rDNA 유전자에 대한 denaturing gradient gel electrophoresis (DGGE)를 수행하였다. 분석된 균주는 개체간의 차이가 있었으나, 대부분 사람의 장내에 살고 있는 normal flora로 동정되었다. 모든 분변에 존재하는 5 개 밴드의 서열 분석 결과에 의하면 Faecalibacterium prausnitzii, Morganella morganii 및 Uncultured bacterium sp.가 나타났고, 항암제 처리 후 Sphingomonas paucimobilis, Lactobacillus gasseri, Parabacteroides distasonis 및 Enterobacter sp.가 증가하였다. 이 연구에서 probiotic으로 알려진 Bifidobacterium과 Lactobacillus를 특이적 PCR primer를 이용하여 동정한 결과, 항암제 투여로 인해 Bifidobacterium과 Lactobacillus의 개체군이 현저하게 줄어들어 diarrhea와 같은 부작용의 원인을 예상하게 하며, 장내 생태계의 주요 박테리아 집단에도 중요한 영향을 미치는 것을 알 수 있었다. 이러한 결과는 항암제 투여와 같이 시간의 흐름에 따른 균총의 변화를 시각적으로 모니터링하기 위하여 PCR-DGGE 분석법이 유용하다는 것을 나타낸다.
Intestinal microbiota is an important factor in the development of immune defense mechanisms in the human body. Treatments with anticancer agents, such as 5-Fluorouracil, Cisplatin, and Oxaliplatin, significantly change the temporal stability and environment of intestinal bacterial flora. The antica...
Intestinal microbiota is an important factor in the development of immune defense mechanisms in the human body. Treatments with anticancer agents, such as 5-Fluorouracil, Cisplatin, and Oxaliplatin, significantly change the temporal stability and environment of intestinal bacterial flora. The anticancer treatment chemotherapy often depresses the immune system and induces side effects, such as diarrhea. This study investigated the effects anticancer agents have on the intestinal microbial ecosystems of patients with gastric cancer. An exploration of the diversity and temporal stability of the dominant bacteria was undertaken using a DGGE with the 16S rDNA gene. Researchers collected stool samples from patients zero, two and eight weeks after the patients started chemotherapy. After the treatment with anticancer agents, the bacteria strains Sphingomonas paucimobilis, Lactobacillus gasseri, Parabacteroides distasonis and Enterobacter sp. increased. This study focused on the survival of the beneficial microorganisms Bifidobacterium and Lactobacillus in the intestines of cancer patients. The administration of antigastric cancer agents significantly decreased Lactobacillus and Bifidobacterium populations and only moderately affected the main bacterial groups in the patients' intestinal ecosystems. The results showed the versatility of a cultivation independent-PCR DGGE analysis regarding the visual monitoring of ecological diversity and anticancer agent-induced changes in patients' complex intestinal microbial ecosystems.
Intestinal microbiota is an important factor in the development of immune defense mechanisms in the human body. Treatments with anticancer agents, such as 5-Fluorouracil, Cisplatin, and Oxaliplatin, significantly change the temporal stability and environment of intestinal bacterial flora. The anticancer treatment chemotherapy often depresses the immune system and induces side effects, such as diarrhea. This study investigated the effects anticancer agents have on the intestinal microbial ecosystems of patients with gastric cancer. An exploration of the diversity and temporal stability of the dominant bacteria was undertaken using a DGGE with the 16S rDNA gene. Researchers collected stool samples from patients zero, two and eight weeks after the patients started chemotherapy. After the treatment with anticancer agents, the bacteria strains Sphingomonas paucimobilis, Lactobacillus gasseri, Parabacteroides distasonis and Enterobacter sp. increased. This study focused on the survival of the beneficial microorganisms Bifidobacterium and Lactobacillus in the intestines of cancer patients. The administration of antigastric cancer agents significantly decreased Lactobacillus and Bifidobacterium populations and only moderately affected the main bacterial groups in the patients' intestinal ecosystems. The results showed the versatility of a cultivation independent-PCR DGGE analysis regarding the visual monitoring of ecological diversity and anticancer agent-induced changes in patients' complex intestinal microbial ecosystems.
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가설 설정
DGGE bands’ profiles were generated for the samples the temporal changes were graphed in Fig. 2. Most microbiota detected in this experiment was intestinal microflora. Characterization of the microflora appears in Table 3.
제안 방법
Amplification of the variable region was accomplished with primers (lacking the GC clamp) and the same thermal cycler (BioRad) program as described above for DGGE. DNA sequencing was performed by the Genotech staff (Genotech co., Daejeon, Korea).
The PCR program consisted of the following steps: initial denaturing at 95°C for 10 min, followed by 30 cycles of denaturing at 95°C for 1 min, primer annealing at 55°C for 1 min, extension at 72°C for 1 min, and final extension for 10 min at 72°C.
The amplification program was 92°C for 2 min followed by 30 cycles of 95°C for 30 sec, 30 sec at the 55°C annealing temperature, and 72°C for 30 sec.
com). The analysis included the number, position, and intensity of PCR-DGGE bands (PCR-amplified 16S rDNA fragments) in the gel.
The current study was designed to investigate the effect of chemotherapy on the intestinal ecosystem of gastric cancer patients. For this purpose, bacterial population fingerprinting of feces was determined using PCR followed by DGGE analysis of the 16S rDNA gene with universal and species-specific primers.
The following PCR program was used: initial denaturation at 94°C for 5 min, three cycles of denaturation at 94°C for 4 sec, annealing at 57°C for 2 min, extension at 72°C for 1 min, 30 cycles of denaturation at 94°C for 20 sec, annealing at 57°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 5 min.
The subjects of this study were 3 patients who were previously diagnosed with gastric cancer in the Gastroenterology Clinic at Pusan National University Hospital in Korea. The patients were given 5-fluorouracil, cisplatin, and oxaliplatin chemotherapy for 8 weeks.
The ward staff collected stool samples from the patients on sampling days –0, 2 and 8 weeks after the start of chemotherapy.
Total DNA was extracted from fecal samples by using the QIAamp® DNA Stool Mini Kit (QIAgen, Canada) according to the manufacturer’s instructions with some modifications [10].
The bands in the profiles represented most of the dominant microbial populations in the community, and their appearance and disappearance reflected approximate temporal changes in the microbial community composition. Using a statistical analysis of each lanes using XLstat program, we observed the change of intestinal microbiota ecosystem, after dosing the anticancer agents just after 2 weeks.
대상 데이터
The subjects of this study were 3 patients who were previously diagnosed with gastric cancer in the Gastroenterology Clinic at Pusan National University Hospital in Korea. The patients were given 5-fluorouracil, cisplatin, and oxaliplatin chemotherapy for 8 weeks.
성능/효과
In conclusion, we demonstrated that PCR-DGGE is a powerful tool for monitoring the effects of chemotherapy administration in gastric cancer patients’ gut ecosystems. The recent development and application of genus-specific primers for intestinal microbes in combination with DGGE expand the potential of this technique for detection of specific populations in some ecosystems.
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