犬隻骨髓間葉幹細胞之病原核酸篩檢

Abstract

近年來犬隻骨髓間葉幹細胞逐漸受到關切，許多研究結果顯示了犬隻骨髓間葉幹細胞在未來臨床獸醫學上應用的可行性。但因單一動物身上取得的骨髓間葉幹細胞數量有限，故必須經由體外細胞培養增加其數量以利臨床醫學的運用。此外，幹細胞在許多疾病上的治療是有時效性的，如使用自體的幹細胞，經過了體外細胞培養以增加數量，常會造成治療的延遲；又因幹細胞本身特性，可進行異體移植而不會造成排斥，如此情形下，若建立犬隻骨髓間葉幹細胞的保存庫，對未來臨床獸醫學的應用應有幫助。 然而，針對幹細胞捐贈動物，目前尚未建立篩檢的準則，以規範幹細胞在未來臨床上使用的安全性。人醫方面目前已有組織與細胞捐贈者的篩檢準則，其中以傳染性疾病為主要的風險考量。因此，參考美國以及歐洲人醫的規範準則，理想上骨髓間葉幹細胞的捐贈犬隻應健康且臨床生理檢查正常，但犬隻仍應進行特定病原的篩檢，以降低嚴重傳染性疾病傳播的風險。 此研究目標是藉由檢測捐贈犬隻血液與骨髓間葉幹細胞中的特定病原核酸，探討特定病原核酸在骨髓來源間葉幹細胞應用上傳播的風險，以協助建立犬隻間葉幹細胞捐贈動物適當且實用的病原篩檢方式。 本研究收集來自動物收容所及動物醫院的犬隻共三十四隻，在安樂後立即採集血液、骨髓液以及骨骺端海綿骨組織，進行間葉幹細胞之分離和繼代，並且對同一隻狗之血液、第二代 (P2) 與第四代 (P4) 的骨髓間葉幹細胞檢體以nested-PCR方式進行病原核酸檢測。本研究主要針對台灣常見且會造成嚴重臨床症狀之傳染性疾病進行檢測，包括: canine distemper、parvovirus、adenovirus、 Babesia canis、Babesia gibsoni、Mycoplasma felis、Mycoplasma haemocanis、Mycoplasma haematoparvum、 Mycoplasma haemomominitum、Ehrlichia canis以及 Anaplasma platys。 結果在十二隻狗以及十三隻狗的血液中分別檢測出canine distemper和parvovirus，而在這些犬隻當中，超過60%的犬隻在P2和/或P4的骨髓間葉幹細胞中仍可檢測出病原核酸。相對的，在血液中被檢測出有Babeisa gibsoni (n=11)、Anaplasma platys (n=1) 、Ehrlichia canis (n=1) 和Mycoplasma haemocanis (n=1) 的捐贈者中，其間葉幹細胞皆未檢測出病原核酸的存在。 基於以上的結果，利用血液作為捐贈者病毒性疾病的篩選準則應為適當的方法，若在捐贈者血液中發現病毒病原核酸，應將此捐贈者剔除。然而，若於捐贈者血液中檢測出Babeisa gibsoni，依據目前研究結果，因病原經過體外細胞培養後無法繼續出現在間葉幹細胞中，此捐贈者的骨髓間葉幹細胞仍可考慮作為臨床應用。

During the past decade, the therapeutic potential of canine mesenchymal stem cells (MSCs) has gradually gained recognition. Various studies have indicated the potential of MSCs for clinical applications in medicine. Although the exact dose of MSCs in clinical application remains unclear, more than several millions of cells at each transplantation site are generally described in the literature. Thus, ex vivo expansion of MSCs is necessary due to its scarce nature to achieve applicable numbers for clinical application. MSCs have also been indicated to be suitable for allogeneic transplantation due to its immunomodulation characteristic. What’s more, allogeneic transplantation is reasonable since many injuries should be treated in a timely manner and autologous transplantation may cause a delay in treatment. Therefore, it is necessary to establish a cryopreservation bank for MSCs for clinical application. However, no guidelines of donor eligibility have been established in veterinary medicine. In human medicine, general guidelines for donor eligibility have been established for donors of cells and tissues, with the risk of infectious diseases transmission being one of the major concerns. According to the Food and Drug Administration (FDA) guidelines in the United States for the eligibility determination for donors of Human Cells, Tissues, and Cellular and Tissue-based products, screening for communicable infectious agents is required prior to donation. Also, in the European Union (EU) Tissues and Cells Directives, similar guidelines are mentioned with additional requirements to exclude donors with systemic infections not yet controlled. Taking these guidelines into consideration, ideally MSCs should be harvested from healthy canine donors with relatively normal physical state. However, pathogen screening should be performed to reduce the risk of severe transmissible infectious diseases. This study aimed to understand the pathogen nucleic acid transmission between blood and isolated mesenchymal stem cells, in order to establish an efficient, effective and practical pathogen screening protocol for donor selection of canine marrow mesenchymal stem cells. Based on the prevalence in Taiwan and the clinical severity of the infectious diseases, pathogens studied in this research included canine distemper virus, parvovirus, adenovirus, Babesia canis, Babesia gibsoni, Mycoplasma felis, Mycoplasma haemocanis, Mycoplasma haematoparvum, Mycoplasma haemomominitum, Ehrlichia canis and Anaplasma platys. Blood, bone marrow and cancellous bone tissue were collected soon after euthanasia from 34 canine donors from animal shelters or animal hospital. MSCs were isolated from bone marrow and/or cancellous bone tissue and ex vivo expanded to 4 passages. Nested-polymerase chain reaction (PCR) was performed on paired blood, second passage (P2) and forth passage (P4) MSCs samples to detect the pathogens mentioned above. Nucleic acid of distemper virus and parvovirus were detected in blood samples from 12 and 13 donors respectively. In over 60% of these donors, nucleic acid of both viruses was further detected in the P2 and/or P4 MSCs. In contrast, in donors with nucleic acid of Babeisa gibsoni (n=11), Anaplasma platys (n=1), Ehrichia canis (n=1), and Mycoplasma haemocanis (n=1) detected in blood samples, the pathogen nucleic acid was not found in MSCs. Based on the results, performing screening tests for viral diseases on blood samples is an appropriate method for donor selection. Due to the high risk of transmitting infectious viral agents into the MSCs, donors with positive blood samples for viral diseases should be excluded. However, since based on the current results, after the ex vivo expansion, the nucleic acid of Babeisa gibsoni no longer existed in the MSCs samples, detecting Babeisa gibsoni in blood sample does not necessarily exclude the donor from being a suitable candidate.