Develvopment of an in vitro cell sheet-based anti-cancer model

Cancer is one of the main causes of death in humans. Research on cancer has been done a lot since the 20th century until the 21st century, but has yet to fully understand it. While the two-dimensional model developed in these situations is used as a standard model for many cancer studies, these two-dimensional models do not reflect the actual cancer environment. Because of these shortcomings, 3D models, similar to the actual environment, are being developed to be applied to patients based on results from the two-dimensional model. However, these three-dimensional models were also totally not considered in the real world of the ECM, which exists in the body, only by using cancer cells to study the form of spheroid, or by mixing different cells. Only recently are these models being developed, and we also developed a cellular sheet-based three-dimensional model with our own technology of cell sheets. The cell sheet is an artificial tissue that mimics the tissue in the body and has been developed to help recover the unrelenting wounds of postoperative patients, burns and diabetes. The research was conducted in the belief that cell sheets could be combined with speroid to create an artificial environment similar to the actual cancer environment. In our study, unlike in the two-dimensional or simple spheroid culture, the presence of the ECM on cell sheets has shown to increase resistance to anticancer drugs in the cancer. Also, in the cellular sheet binding model of transversed insurer cells, the cell sheet model could be seen spreading around the invoice, unlike the former models. In addition, it was possible to observe the hypoxia area inside one of the main characteristics of cancer in the existing three-dimensional models. The EMT-related representative molecules like vimentin, TGF-β1, N-cadherin were analyzed through RT-qPCR and Western blot, and the results were shown that three molecules increased. Furthermore, stability could not be established in a traditional two-dimensional model. However, stability of a three-dimensional model created using cancer tissue from patients could be maintained up to 30 days. In addition, through the transduction of GFP into tissue, live area can be confirmed with fluorescence in real time. This allowed us to identify the impact on cancer tissues over time in a five-day anti-cancer reaction. This is expected to help select an anti-cancer drug for each of patient. | 암은 인간의 주요 죽음의 원인 중 하나이다. 암에 대한 연구는 20세기를 넘어 21세기인 현재까지 많이 진행되었지만 아직 암에 대하여 완전히 이해하지 못 하고 있다. 이러한 상황에서 개발된 2차원 모델은 많은 암 연구에 있어서 표준적인 모델으로서 사용되고 있지만, 이러한 2차원 모델은 실제 암의 환경을 반영하지 못하는 단점이 있다. 이러한 단점으로 인해 2차원 모델로 부터의 결과를 토대로 환자에게 적용하기엔 무리가 따르고 이를 극복하기 위해 실제 환경과 유사한 3D 모델들이 개발되고 있다. 하지만, 이러한 3차원 모델 또한 단지 암 세포를 가지고 spheroid라는 형태를 만들어 연구하는 방식이거나 다른 세포를 섞어 spheroid를 만들어 연구하는 형태를 띄고 있을 뿐, 실제 체내에서 존재하는 ECM 부분은 전혀 고려되고 있지 않은 상황이었다. 최근에 들어서야 이러한 부분들이 고려된 모델들이 개발되고 있고, 우리 또한 세포시트라는 자체 기술을 가지고 이러한 기류에 편승하여 세포시트 기반의 3차원 모델을 개발 하였다. 세포시트는 체내의 조직을 흉내 낸 인공조직으로 수술 후 발생하는 결손 부위, 화상, 당뇨환자의 아물지 않는 상처의 회복을 돕기 위해 개발 되었다. 세포시트를 spheroid와 결합하여 실제 암 환경과 유사한 환경을 인공적으로 만들 수 있다고 생각하여 연구를 진행 하였다. 우리의 연구 결과에서, 2차원 또는 단순 spheroid culture에서와는 다르게 세포시트의 ECM의 존재는 cancer에 항암제에 대한 저항성을 더 높여주는 모습을 보여 주었다. 또한, GFP가 transfection된 cancer cell로 만든 세포시트 결합 모델에서, 전자의 기존 모델들과는 다르게 세포시트 모델은 invasion과 주변으로의 퍼져나가는 모습을 확인 할 수 있었다. 또한, 기존의 3차원 모델들에서 관찰 할 수 있는 암의 주요 특징 중 하나인 내부의 hypoxia 지역을 관찰 할 수 있었다. EMT 관련 대표 분자인 vimentin, TGF-β1, N-cadherin을 RT-qPCR과 western blot을 통해 분석한 결과 vimentin과 N-cadherin, active TGF-β1은 증가하였고, N-cadherin은 감소하는 결과를 보여 나타내었다. 더 나아가 환자로부터 얻은 암 조직을 이용하여 만든 3차원 모델에서 기존 2차원 조직 모델에서 확립할 수 없었던 안정성을 30일 까지 확보 할 수 있었다. 또한 조직으로 GFP 도입을 통해 살아있는 부분을 형광현미경으로 실시간으로 확인 할 수 있었다. 이러한 점 5일간의 항암제 처리 반응에서 시간에 따른 암 조직에 대한 영향을 확인 할 수 있었다. 이것은 각 환자에 맞는 항암제 선정에 도움이 될 것으로 예상한다.Maste

Development of EBV-encoded small RNA targeted PCR to classify EBV positive diffuse large B-cell lymphoma (DLBCL) of the elderly

의생명과학전공/석사광범위큰B세포림프종(diffuse large B-cell lymphoma, DLBCL)의 분류가 최근 더욱 세분화되어 2008년 개정된 World Health Organization (WHO) 분류에서는 Epstein-Barr virus (EBV) positive DLBCL of the elderly가 처음으로 포함되었다. 이 질환은 50세 이상의 나이든 사람에게서 발생하며 EBV와의 밀접한 연관성이 확인되어 새롭게 분류되었다. 이에 본 연구에서는 2002년1월부터 2012년12월까지 수원시에 소재하는 가톨릭대학교 성빈센트병원에서 DLBCL로 진단 받은 91명을 대상으로 표준 검사법으로 알려진 Epstein-Barr virus-encoded small RNA-1(EBER1)-in situ hybridization과 분자생물학적 방법인 DNA polymerase 유전자를 표적으로 하는 nPCR (nested polymerase chain reaction)과 EBER 유전자를 표적으로 하는 PCR (polymerase chain reaction)을 통하여 Epstein-Barr virus (EBV)의 검출율을 비교 하고, EBV positive DLBCL of the elderly로 분류되는 예가 얼마나 되는지 확인하고자 하였다. 그 결과 EBER 1-ISH에서 3건 (3.30%), nPCR에서 26건 (28.57%), EBER PCR 에서 3건 (3.30%)의 EBV양성 결과를 얻었다. EBER-ISH와 PCR의 결과에서 양성은 3건으로 동일한 결과를 얻었으며, 염기서열 분석을 실시하여 PCR에서 양성으로 나온 예가 모두 EBV의 EBER 유전자인 것을 확인할 수 있었다. 총 91명의 DLBCL 환자 중 EBV positive DLBCL of the elderly 해당하는 환자는 2명으로, 발병 장기는 lymph node에서 1건, extra-lymph node에서 1건으로 나타났다. 이와 같은 결과는 지금까지 보고된 아시아 지역에서 EBV positive DLBCL of the elderly로 분류되는 비율 5~11%에 비해 낮은 빈도로 나타났고, 서양지역에서의 5% 이하로 보고되는 결과와는 유사하였다. 아직 국내에서 EBV positive DLBCL of the elderly 에 대한 보고가 많이 없어 진단의 기준이 아직까지도 모호한 실정이다. 본 연구는 수원 지역에서의 EBV positive DLBCL in elderly 분류에 관련된 첫 보고서로서, EBER1-ISH 결과와 PCR 결과를 토대로 EBV positive DLBCL of the elderly의 분류를 시도하였다.restrictio

생쥐 난소조직을 이용한 가임력보존 방법에 관한 연구

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의학과 분자유전체의학전공, 2016. 2. 서창석.Introduction: As the diagnosis and treatment of cancer have been dramatically improved, fertility preservation in female cancer patients has been up in light to improve their quality of life. Oocyte banking and embryo cryopreservation commonly used as fertility preservation options, however, they cannot be applied to pre-pubertal girls and unmarried women. Fertility preservation with ovarian tissue (OT) can provide alternative instead of oocyte and embryo while there are major hurdles to enhance the efficiency of the procedures. First, cryoinjury is usually occurred during cryopreservation process. Second, ischemic injury is also spontaneously happened until re-vascularization, such as 2 days and 5 days are needed to initiate the re-vascularization. Finally, OT has a risk to re-implant the malignant cells after transplantation. This study was aimed to 1) compare the deleterious effects of cryoinjury and ischemic injury on the quality of OT after cryopreservation and transplantation process 2) decrease the cryoinjury after vitrification-warming process using several types of antifreeze proteins 3) diminish the ischemic injury via enhancement of re-vascularization after auto-transplantation 4) optimize the two-dimensional follicle culture system using mouse model. Methods: For the comparison of cryoinjury and ischemic injury (Exp I), a total of 160 ovaries were harvested from 6-week-old female B6D2F1 mice. Ovaries were randomly divided into eight different groups consisting of two control groups (fresh and vitri-con) and six experimental groups according to the presence or absence of vitrification and transplantation. (fresh OT [FrOT]-day [D] 2, FrOT-D7, FrOT-D21, vitrified OT [VtOT]-D2, VtOT-D7, and VtOT-D21). In the fresh control group, OT was fixed immediately after ovariectomy, and in the vitri-con group, OT was fixed after the vitrification-warming procedure. All six experimental groups were auto-transplanted with fresh or vitrified-warmed OT, and then the mice were sacrificed by cervical dislocation at 2, 7, or 21 days after grafting. To investigate the detrimental impacts of these injuries, histology, cell-death, blood vessel distribution in OT and ELISA for FSH level For decrease cryoinjury during vitrification-warming process (Exp II.), a total of 140 mice were sacrificed to collect sexually mature ovaries from 6-week-old aged female B6D2F1 mice. In Exp II-I, a total of 240 whole ovaries were randomly distributed to one of three groups: the fresh control group, the vitrification control group, or the AFP-treated group. The AFP-treated group was further divided into nine subgroups according to AFP type (e.g., FfIBP, LeIBP, and type III AFP) and dose (0.1, 1.0, and 10 mg/mL). After two-step vitrification and four-step warming process, the quality of ovary was assessed. Then, auto-transplantation of ovary was carried out to determine whether the cryoprotective effects of LeIBP could also be seen in OT after transplantation (Exp II-II). A total of 20 B6D2F1 mice were randomly divided into two groups: one group that received 10mg/ml of LeIBP-treated ovaries and another that received the vitrification control ovaries. We used only 10 mg/ml of LeIBP-treated group for this experiment because this group showed the best results in Experiment II-I. The quality was evaluated by histology, TUNEL, immunohistochemistry and serum FSH level on each evaluation days (Day 2, 7 and 21 days after transplantation). Next, a combination of simvastatin and/or methylprednisolone was treated to diminish of ischemic injury during avascular period after transplantation (Exp III). Following comparison study, the mice were treated with 5 mg/kg of simvastatin and/or methylprednisolone 2 h before ovareictomy and then the ovaries were cryopreserved by two-step vitrification process as previously described in Exp I. One week later, vitrified OTs were warmed by four-step warming process and then auto-transplanted under bilateral kidney capsules. Similar to the Exp II-II, the mice were sacrificed by cervical dislocation on 2nd, 7th or 21st of the transplantation period to assess the quality of OT. Macroscopic and microscopic examination, immunohistochemistry for blood vessel, flow cytometry for CD45, serum AMH ELISA were carried out on each evaluation days. Moreover, oocyte retrieval from graft and further in vitro fertilization were also performed to evaluate the drug safety on gametogenesis and embryogenesis. Finally, Pre-antral follicles were mechanically isolated from 2-week-old BDF-1 mice and randomly assigned into two groups according to the culture methods (with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layer with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layerwith or without oil layerwith or without oil layer with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layer , Exp IV). In vivo matured oocytes were collected using superovulation to compare the growth, cytoplasmic normality, gene expression and embryonic development. Ovarian follicles were in vitro cultured for 10 days and cumulus-oocyte complexes were harvested at 16-18 hours after hCG and EGF treatment. Mature oocytes were assessed their maturational ability and developmental competence in vitro. Results: In Exp I, The vitrification-warming procedure decreased the intact (grade 1, G1) follicle ratio in the vitri-con and FrOT-D2 groups compared with that in the fresh control, and this ratio was reduced more by ischemic injury after transplantation (fresh: 64.2%, vitri-con: 50.3%, and FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovarian tissue than in the fresh control, and it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0%, and FrOT-D2: 26.8%). The mean number of follicles per section and CD31-positive area was significantly reduced after vitrification and transplantation. (the number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, and FrOT-D2: 17.9 ± 2.1CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9%, and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1%, and FrOT-D21: 70.7%CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6%, and FrOT-D21: 7.5 ± 0.8%). In Exp II-I, the percentage of grade 1 total follicles was significantly higher in only the 10 mg/mL LeIBP group than in the vitrification control while all of AFP-treated groups had significantly improved grade 1 primordial follicle ratio compared with the vitrification control. The apoptotic (TUNEL-positive) follicle ratio was significantly decreased in the 1 and 10 mg/ml of LeIBP treated groups. The proportion of τH2AX positive follicles was significantly reduced in all AFP-treated groups while the Rad51-positive follicle ratio was significantly decreased in only FfIBP and LeIBP treated groups. In ExpII-II, after auto-transplantation of OT vitrified with 10 mg/ml of LeIBP, the percentage of total Grade 1 follicles and primordial Grade 1 follicles, the extent of the CD31-positive area were significantly increased. Moreover, the level of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated group than in the control group. In Exp III, The group that received simvastatin and methylprednisolone showed a significantly improved intact (G1) follicle ratio (D2: p<0.001, D7: p<0.05 and D21: p<0.001), apoptotic follicle ratio (D21: p<0.05), CD31-positive area (D7: p<0.05 and D21: p<0.05), and serum AMH level (D7: p<0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rate, the number of total and apoptotic blastomere per blastocyst and ICN/TE ratio among the four transplantation groups. In Exp IV, With respect to the follicular growth, the diameter of follicles in oil layer culture was significantly higher than that of without oil layer. In addition, maturational criteria including survival in oil layer, pseudo-antral like cavity formation, ovulation and oocyte maturation, also significantly increased compared with the without oil layer. Late stage of culture, estradiol on D10 and progesterone on D11 in spent medium of oil layer was statistically significant different between without oil layer culture condition. When comparing the mRNA expression in matured oocytes, no significant difference was observed between in vivo and in vitro derived oocytes. On the other hand, in vitro grown and matured oocytes increased the level of reactive oxygen species and decreased the mitochondrial activity when compared with the in vivo mature oocytes with statistical significance. Moreover, cortical granules of both in vitro derived oocytes seemed to be more clumped and unevenly distributed than in vivo control. However, no significant difference was noted in actin filament configuration and spindle normality between in vitro and in vivo derived oocytes. Conclusions: Cryoinjury and ischemic injury are main cause of follicular depletion during fertility preservation process using ovarian tissue. Inevitable post-transplantation ischemia seems to be more deleterious than cryoinjury during cryopreservation process. Then, cryoinjury and ischemic injury could be decreased by use of AFPs and a combination of simvastatin and methylprednisolone. It can improve the quality of OT via promotion of vessel integrity in OT after cryopreservation and transplantation process. Therefore, minimizing cryoinjury and ischemic injury by enhancing vascularization is needed to improve the ovarian function after fertility preservation. Finally, optimization of two-dimensional in vitro follicle culture method was also attempted to avoid the re-implantation of residual malignancy cells in OT after transplantation and found the cause of reduction in embryonic development competence from in vitro derived oocytes.INTRODUCTION 1 MATERIALS AND METHODS 7 RESULTS 39 DISCUSSIONS 93 ABSTRACT IN KOREAN 129Docto