Grant amount：\4680000 （ Direct Cost: \3600000 、 Indirect Cost：\1080000 ）

In the present study, we aimed to investigate the therapeutic effect of stem cell administration by creating a diaphragm hernia model by administering nitrophen to maternal mice.We tried to make a model mouse by orally administering 25 mg of nitrophen to a mouse on 8.5 days of pregnancy. Although some effects of the drug were observed, such as a decrease in fetal body weight, it was confirmed that the incidence of diaphragmatic hernia in fetuses was about 10% of the total, which was extremely small.

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Grant amount：\47450000 （ Direct Cost: \36500000 、 Indirect Cost：\10950000 ）

Among the above-mentioned disease groups, hypoganglionosis (HYPO), which has a poor prognosis, was focused first. HYPO is characterized by the presence of intestinal ganglion cells, but the number is extremely small. Similarly, a mouse (JF1) in which a decrease in intestinal nerve cells was used to elucidate the effect of stem cells from human exfoliated deciduous teeth (SHED). As a result, JF1 mice gained weight , indicating the effectiveness of SHED. Next, the characteristics of SHED derived from HYPO patients (HYPO-SHED) was analyzed. SHED is one of a mesenchymal stem cell (MSC), and HYPO-SHED has a phenotype consistent with MSC, and analysis was conducted on its proliferative potential and tumorigenity.

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Grant amount：\4680000 （ Direct Cost: \3600000 、 Indirect Cost：\1080000 ）

There are three fractions (L1,L2,L3) in AFP (Alpha-feto protein) which is a typical tumor marker. Purpose of this project is to separate L2 fraction from L3 using an immunology and mass spectrography, and establish non-invasive simple diagnostic system.
By immuno electrophoresis method using serum and tissue samples obtained from patients (target) with solid malignant tumors and benign diseases (control), physiological regression of L2 fraction was developed

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Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）

X-ray examinations have become essential in medical facilities. In particular, chest radiography is a frequently performed examination, which causes patient exposure. Children have higher radiation sensitivity than adults, and it is necessary to set appropriate imaging conditions and to evaluate exposure dose at the time of examination. In this study, we developed a polygon phantom that can be output by 3D printer for children with variable system from CT volume data, and evaluated the method of evaluating organ absorbed dose by Monte Carlo simulation, and clarified the printing condition of phantom that is 3D printed out close to the real body.

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Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）

For the adrenal gland neuroblastoma, a three-dimensional organ model was created from a preoperative photographed CT image using a 3D printer. Using a resin that can be inserted into a trocar, a body was created, and it was used for simulation of tumor position in laparoscopic view, and examination of the best position of the trocar. We also modeled adrenal gland and liver model for liver metastasis cases of neuroblastoma and conducted surgical simulation.
For hepatoblastoma, the same pediatric malignant tumor as neuroblastoma, a three-dimensional organ model was created from preoperative CT images. We confirmed the positional relationship between the portal vein and the hepatic vein and the position of the tumor, and simulated the resection line before surgery. We found the utility such as being able to more easily understand the anatomical information.

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Grant amount：\4680000 （ Direct Cost: \3600000 、 Indirect Cost：\1080000 ）

The aim of this study was to develop a model of nursing assistance and problem solving to improve the quality of life of children and parents from school age to adolescence after liver transplantation. Child and family monitoring indicators were developed based on the results of a survey of children and parents, interviews, and a review of the literature. The survey results and literature review led to a conceptual framework, nursing assistance model, and pattern-specific nursing assistance guidelines for improving the quality of life of children and parents from late school age to adolescence after living donor liver transplantation. The developed nursing assistance model was practiced, evaluated, and revised, and the content was refined to construct a nursing assistance model for improving the quality of life of children and parents from late school age to adolescence after liver transplantation.

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Grant amount：\5070000 （ Direct Cost: \3900000 、 Indirect Cost：\1170000 ）

This study is aimed at revealing need for the grief care and planning a further support program. Through interviews to the parents losing their kids, following things seem common and often-heard opinions; “We don’t want to think he/she (kids) never exist from the very beginning.” “Wanna you to know and understand him/her.” “Want a place where I am able to explain myself.”
In this grief care program, four times of group interviews and a single event ware done. In addition, a support book was also prepared.

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Grant amount：\4940000 （ Direct Cost: \3800000 、 Indirect Cost：\1140000 ）

Pediatric malignant soft tissue tumor has a poor prognosis, and a new therapeutic target is desired. Currently, Forkhead box M1 (FOXM1) is one of the most notable molecules as a new therapeutic target in major malignant tumors.
We evaluated FOXM1 expression in 92 patients with rhabdomyosarcoma (RMS) and 106 synovial sarcoma (SS) among pediatric malignant soft tissue tumors. We showed that FOXM1 expression is associated with poor prognosis. In RMS, FOXM1 correlated with VEGF expression and angiogenesis. In cDNA microarray analysis of SS cases, we showed that cell cycle related gene expression correlates with FOXM1 expression. We showed that FOM1 inhibition could be a new treatment option in studies of cell lines of each tumor.

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Grant amount：\44720000 （ Direct Cost: \34400000 、 Indirect Cost：\10320000 ）

The aim of this project is to develop a novel non-invasive diagnostic methods and therapeutic strategy for pediatric cancer, which can be translated into the upcoming clinical trial. We have identified 1) potent serum-based biomarkers for the therapeutic stratification of neuroblastoma and rhabdomyosarcoma, 2)a potent delivery system using human mesenchymal stem cells targeting neuroblastoma, 3) a novel immunotherapeuric strategy for pediatric cancers using peptide vaccine system, anti PD-1 antibody, or NKT cell therapy, 4) novel molecular-based stratification system for pediatric cancers detected by next generation sequencing analysis.

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Grant amount：\5330000 （ Direct Cost: \4100000 、 Indirect Cost：\1230000 ）

We used the MYCN transgenic mouse which was a neuroblastoma model mouse for determining the possibility of the new molecular target in the neuroblastoma. The tumor samples were obtained from homo and hetero MYCN transgenic mouse. The pathological examination was performed for homo and hetero MYCN transgenic mouse samples. And Hh signal proteins (Sonic Hh, GLi1, Patched) were dertermined using immunostaining method. H.E. stains of both samples were shown undifferentiated subtype of neuroblastoma and there were not differences between both groups. Sonic Hh, Patched was strong positive, but the GLi1 was negative in both group. From the results, there was not difference between two groups, even if the survival rate of each group were different. In the human neuroblastoma specimen, the GLi1 was strongly positive. However, in MYCN transgenic mouse, GLi1 was negative. Therefore the Hh signal activation was different between human neuroblastoma and MYCN trancegenic mouse.

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Grant amount：\5590000 （ Direct Cost: \4300000 、 Indirect Cost：\1290000 ）

The objective is to get suggestion to construct the optimal developmental care for premature infants in the NICU. We evaluate their quality of sleep, sleep-wake rhythm and revealed the characteristics of physiological daily variation. In addition, we carried out interventional observation for simulated intrauterine environment. As a result, we recognized decline trend in amount of activity within dark NICU environment (<50Lux). There are no clear differences in quality of sleep. By the frequency analysis of the noise, infants cried more when noise are above 70-80dB. This result meant that noise stimulate infants to awake rather than light.

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Grant amount：\4420000 （ Direct Cost: \3400000 、 Indirect Cost：\1020000 ）

This study was Action Research for exploration the possibility of Children's Hospice in Japan. The purpose of this research was to clarify the experiences of children with life threatening illness and their parents, and their palliative care needs. Palliative care needs of them are needs for their development. Especially, QOL in infancy and after graduate high school were decreasing. We have concerned with three cases of children in terminal stage and found not only medical care but also the necessity of children's and family developmental support. Next, we made some citizen forum and meetings as enlighten programs for pediatric palliative care (children's hospice), and some programs to support the children's and the family's QOL. Finally, we made a network for children's hospice through cooperating with not only heath care providers but also ordinary people and people in multiple ranges. Through the process of action research, a direction for children's hospice in Japan were showed.

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Grant amount：\4420000 （ Direct Cost: \3400000 、 Indirect Cost：\1020000 ）

【Purpose】The purpose of this study is to analyze the usefulness of Glypican3 as a novel diagnostic tumor marker for pediatric solid malignant tumor, and to establish the new diagnostic system. Final goal is to develop the immuno-genetic therapy targeted for the Glypican3.【Method】The immunohistochemical analysis and serological analysis of Glypican3 and AFP was performed for the pathological materials and patient’s serum. 【Results】Immunohistochemlically and serologically, most cases of hepatoblastoma and malignant germ cell tumor showed positive for Glypican3, and one of thirds Wilms tumor, alveolar type rhabdomyosarcoma and undifferentiated sarcoma were also positive. Concerning AFP, most cases of hepatoblastoma and malignant germ cell tumor showed positive. Concerning non-neoplastic patients’, serum Glypican 3 was high just after birth and gradually decreased according to their age. The level normalized by the 1 year.【Discussion】Glypican3 was confirmed as the novel diagnostic marker for pediatric solid malignant tumors. Based on this research, for the refractory cases of pediatric solid malignant tumor over 1 year, such new treatment immuno-genetic therapy targeted forGlypican 3 will be expected.

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Grant amount：\17290000 （ Direct Cost: \13300000 、 Indirect Cost：\3990000 ）

Intrapulmonary mesenchymal stem cells (MSC) were administered to the rat with nitrophen-induced diaphragmatic hernia. As a result, PCNA positive cells are increased.DNA was extracted from the 20 patients and parents of congenital diaphragmatic hernia. The microdeletion of TCTE3 was detected. This might be the responsible gene for congenital diaphragmatic hernia.Abdominal muscle flap method was performed for the reconstructive surgery in 11 cases with recurrent diaphragmatic hernia. All of the patients showed good outcome.Liver-like cells were induced from SHED. We are just starting to make the sheet of the striated muscle cells derived from SHED.

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Grant amount：\4550000 （ Direct Cost: \3500000 、 Indirect Cost：\1050000 ）

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Grant amount：\4420000 （ Direct Cost: \3400000 、 Indirect Cost：\1020000 ）

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Grant amount：\4040000 （ Direct Cost: \3500000 、 Indirect Cost：\540000 ）

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Grant amount：\3500000 （ Direct Cost: \3500000 ）

We compared gene expressions between favorable and unfavorable neuroblastomas by microarray system, and it showed about 400 of unfavorable tumor dominant genes and about 40 of favorable tumor dominant genes. A high expression of neuronatin (Nnat) gene in favorable neuroblastomas were found. Neuronatin is a gene which is considered to relate to the neurogenesis, and the expression appears in nervous systems from the hindbrain to the peripherals during the prenatal periods. It is an imprinted gene, highly conserved in mammalian spices. The neuronatin mRNA expression was investigated in 70 of neuroblastoma samples by quantitative RT-PCR, and the expression was significantly high in favorable groups than unfavorable groups. Although the detailed function is not revealed, the high expressions of neuronatin were found to be associated with good prognoses in neuroblastoma, which might indicate the tumor differentiation, and its expressions in unfavorable tumors were supposed to be epigenetically suppressed.

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Grant amount：\14500000 （ Direct Cost: \14500000 ）

Insulin-like growth factors (IGFs) are one of the major growth factors related to lung development. The purpose of this study is to analyze the effect of IGF using organ cultured lungs.
The lungs were dissected from E11 to E18 mice. Total RNA was then extracted from those lungs. The expression of IGF and its receptors was analyzed by RT-PCR.
The lungs dissected from E17 mice were cultured in the BGjb medium for 48 hours. The lungs were divided into the following three groups ; the lungs were cultured only by the medium (group I), or the medium containing either IGF-1 (group II) or IGF-2 (group III). The expressions of TTF-1,Sp-C, and alpha-SMA were analyzed by both RT-PCR and immunohistochemistry.
The m-RNA expression of IGF-1 receptor was observed to be higher from E16 to E18 than at any other embryonic stages. TTF-1,Sp-C and alpha-SMA were more highly expressed in groups II and III than in those of group I. TTF-1,Sp-C, and alpha-SMA positive cells were increased in group II and III compared to group I.
Based on our findings, IGF is therefore suggested to induce alveolar maturation in the late stages of fetal lung development. IGF is considered to be one of the candidates for promoting lung development in patients with congenital diaphragmatic hernia.

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Grant amount：\3400000 （ Direct Cost: \3400000 ）

β-カテニンのoncogenesisへの関与は小児悪性腫瘍においては近年、肝芽腫の発生に強く関与する報告がなされている。肝芽種の約50%の症例においてβ-カテニンの変異が検出されており、その発生への関与が示唆されている。しかし他の小児固形悪性腫瘍へのβ-カテニンの関与は今のところ不明である。ウイルムス腫瘍の発生にはWT1遺伝子異常が関与するとされてきたが、その頻度は10%程度であり、oncogenesisにどのように関わっているかの詳細は未だ不明である。近年、β-カテニンもウイルムス腫瘍に15%程度の頻度で異常を認めるとされており、本研究ではβ-カテニン遺伝子とWT1遺伝子のウイルムス腫瘍の発生ならびに発育における関与を明らかにすることを目的とした。1964年より2005年の間に九州大学小児外科教室において経験されたウィルムス腫瘍40例を対象とし、免疫組織学的にβ-カテニン、WT1遺伝子の発現を検討した。β-カテニンについては細胞膜に陽性を示したものが38例(100.0%)、細胞質に陽性を示したものが35例(92.1%)、核内移行を示したものが9例(23.7%)であった。組織型別での陽性率は腎芽型成分に35例(92.1%)、上皮型細胞成分に12例(31.6%)、問葉型成分には2例(5.3%)の陽性率であった。WT1については細胞質に陽性を示したものが30例(79.0%)、核内移行を示したものが22例(57.9%)であり、組織型別での陽性率は、腎芽型細胞成分に21例(55.2%)、上皮型細胞成分に14例(36.8%)、間葉成分には3例(7.9%)の陽性率であった。β-カテニンにて核内移行を示した9例は全例WT1にても核内移行を示した。さらに組織内におけるDNAの発現と変異を調べるためにMicrodissectionにてこれらのパラフィン標本より組織型別にDNAを抽出し、検出されたDNAをSequencerを用いてdirect sequenceを行った。しかしqualityの高いDNAを得ることができず、有意な結果を得ることはできなかった。以上よりウィルムス腫瘍におけるβ-カテニン、WT1遺伝子のoncogenesisへの関与に関しては免疫組織学的検討により同時に核内移行を示す約20%の症例において何らかの関与があることが示唆された。

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Grant amount：\3400000 （ Direct Cost: \3400000 ）

乳児神経芽腫マススクリーニングによる発見症例は、その生存率は98%と非常に高く、一見その目的は達成されているようにみえるが、マス検査陰性後に臨床的に発見される症例(マス陰性例)が数多く存在し、それらは進行症例が多く、予後不良である(J Pesiatr Surg.1998:33,1674-1678)。日本以外で全国規模でマスを施行している国は世界になく、多数のマス発見例及びマス陰性例の詳細な生物学的特性の解析と臨床経過の検討は、マスの有効性の評価及びマス症例の治療方針の決定に直結するだけでなく、神経芽腫全体の生物学的多様性を究明するデータとなることが予想される。
現在までの研究で、神経芽腫の遺伝子異常を高感度かつ迅速に解析を行う手法としてMYCN amp.と1p del.に関してflourescence in situ hybridization(FISH)による解析を確立し(J Pediatr Surg,1999:34,1615-1619)、また、MYCN amp.と17q gainに関してReal Time PCR(TaqMan^<TM>PCR)による解析を確立した(Cancer Lett.2001:10,89-94)。
MYCN amp.に関しては、今年度の研究によりFISHとReal Time PCRによる組み合わせが最も正確にamplificationの状態を評価することができて、神経芽腫の悪性度と非常に相関している結果を得て発表した(J Pediatr Surg.2004:39,63-68)。
さらに、定量的PCRによりMYCN amp.,17 q gain,1p del.,11p del.,14q del.の同時解析を行った結果、MUCN amp.,17q gain,1p delの高感度解析の組み合わせにより、乳児神経芽腫の生物学的悪性度の層別化が可能であり、マス休止後の臨床的に発見される乳児神経芽腫の治療方針に決定に有意義であると考えられた。

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